Some possibilities:

Resurrection could age the person involved so that they would die sooner (say 10 years for humans and scaled to the lifespan of other races) from natural causes, multiple Resurrections could lead to a person that dies again only days after being raised.

Resurrection could have a chance to fail (say 12.5%), making the person forever dead and with each subsequent resurrection doubling the chance of failure.

Resurrection works but is not perfect and the subject is weakened by it. Eg: the person loses ability scores (say -2 con and one other ability score at random).

Resurrection works but the costs increase dramatically each time. 2nd is 10 times more expensive, 3rd is 100 etc.

Or resurrection works as normal but the body must be whole. All you need to do to make an assassination stick is to behead the person and take the head with you or burn the body.

There's two obvious loopholes in the spell that don't require any DM fuckery.

The first is that the soul of the target must be "free and willing".

The willing part is very important. Who says anyone wants to be brought back? There's a bunch of afterlife options in d&d, but many of those are quite nice. If a soul, no matter how its body died, is happy and loving where they are, they can just refuse the call of resurrection .

The other canon option is about the "free" part. There are things (demons, and others) that can entrap a soul. It would take fairly little for that to become known to players who might then arrange for such to happen.

That's just the spell itself, no need to have ways that the resurrection spell casting is blocked unless you want it to be other reasons.

Other answers have already covered the best options in that regard; deific intervention being the most powerful option. A god with an objection to this empire could make it difficult to impossible for anyone to be resurrected, even if it came down to making deals with gods of death and the afterlife.

I particularly liked the idea of a weapon that kills, turns the victim undead, and then kills the undead. It isn't technically going to bypass resurrection by RAW, but it isn't a stretch to do DM's option of world building to make it RAW for your setting.

But you can go the route of the spell itself being blocked. Counter magic exists. The weapon that another comment suggested for turning them undead could instead just lay a counter to resurrection. Something that uses the same mechanics as countering a spell.

You think Henchmen #4 is getting the royal treatment after he died falling down the stairs cause his helmet was on backwards? Fuhgetaboutit. You want to take a shot at the king? You'd better not miss (and make it so he can't get back up). They shouldn't worry about cutting through the rabble as they wouldn't warrant such special treatment. A good assassination plot would include measures to ensure the target stays dead. Even True Resurrection requires the soul to be free and willing. Soul can't be free if it was put in a soul cage. Perhaps they made a deal with the devil at some point with their soul on the line.

Just random suggestions:

Normally that's exactly how it works. But then, someone important gets killed, and nothing can be done to resurrect them. No spells, no deity intervention. They're just gone and their corpse is there.

Kinda weird, but lots of stuff happens, things are busy. Then a month later it happens again. And the guy it happened to is important (captain of the city guard / head of the museum? / head of the wizard school). It's enough that it's genuinely weird and people get concerned.

The third time, they intercept the assassin. Depending on how right the party plays their cards, maybe they're able to capture and interrogate the person, or there's a particularly weird magical artifact.

The fourth time it's one of the party that's being targeted. Maybe have for-real death with no resurrection as the consequence at hand to give the adventure stakes as things ramp up.

Etc etc, you can see where it's going. The "explanation" could be a few different things, but I actually wouldn't make it clear to the party right away. Maybe the people are being kidnapped, and perfect fake-corpses through some magical means are left behind. Maybe they're on another plane. Maybe it's just some permadeath spell that someone researched in the fantasy-Manhattan-project of the empire your party's going up against, and part of the climax is them destroying the spell because some things are better left unknown.

Etc etc. Something along those lines is how I would handle it and then just see how it plays out. Hopefully this is useful.

This may not work with the campaign you want to run but having resurrection work very well, to the point that there is starting to be an uprising of the "common folk" for a change. That because of this spell there is a small but growing distaste for the ruling class and its starting show up in altercations and small fights that could easily transition into something bigger.

I think deities are an option. Perhaps a non-mortal being won’t permit the healing/resurrection of a monarch for one arbitrary reason or another. It could be that the assassin is ordained by said deity.

Or maybe something that happened to the person at their death or after death made the one who died want to stay dead? The soul’s willingness to return to the body is a key requirement you can use for lore around resurrection.

Just a couple of thoughts.

I'm just a weirdo who just likes dnd memes but doesn't actually play, so no idea if this is possible... but could you have some kind of magic item or potion that causes extremely rapid aging? Like the guy who choose poorly in Indiana Jones? True Resurrection doesn't work on someone who died of old age.

Or kidnap them and have a ghost Horrifying Visage them until they die of old age?

I would personally go with a magical mcguffin. My first thoughts would be a magic dagger. Maybe a small "ceremonial" pendant type. Discovering it, could be a useful plot device, both in that it exists (and so a marker for high level assassins) and how it works.

As for how it works, as an idea, when stabbed into a dead body, it does something nasty/scary to the body. E.g. it contains a drop of vampire blood, and a tiny vial of holy water. The body is raised as a vampire, and then immediately killed by the holy water. This breaks the link needed for resurrection. It could also contain a single drop of the essence of a god of death. Resurrection simply won't work on anyone "infected". This could be particularly scary if an assassin uses his dying act to stab a PC in the leg. He's not dead, but now can never be resurrected.

There's a lot of mileage in investigating the source of the mcguffins. Do they have the body of a dead god? Are they holding a vampire lord captive (or led by one)? Is it a mercenary cult, or a core political power?

I've been plotting out some ideas for what is essentially the inverse of your campaign for some months now. My players will be an elite group of saboteurs and seditionists sent to the capital city of some evil empire in order to bring it down from the inside, however best they see fit.

I ran into the same problem you did. If this empire is ostensibly so powerful, wealthy, and influential, then why couldn't they just resurrect anyone important?

I realise it's not quite as exciting as the other answers, but ultimately I did decide that I'll just heavily restrict the resurrection spells. Resurrection will be theoretically possible, but in the setting, the only known way of accomplishing it is with a powerful and limited-use artifact locked deep away within the empire's vaults. Destroying it or stealing it would be a very good avenue for the players to pursue in order to destabilise the establishment.

On the players' side, should I ever actually get the campaign off the ground, I'll tell them that resurrection spells will not be generally allowed to pick for level ups. I think I would be ok with them spending significant time and effort, maybe a chain of quests, to discover the spells themselves if that's what they're truly interested in and think will advance their cause, but with the warning that if word gets out it will paint a massive target on their backs since that kind of power could completely reshape the worldwide political landscape.

Hopefully, in addition to solving the problem of making sure their high-profile assassinations actually stick, this policy would also encourage the players to play a bit cautiously and generally keep the stakes high.

Anyway, all this to say, shaping the rules and lore of your world to more precisely fit the experience you are trying to provide makes a lot of sense to me, and is worth considering. As long as you get buy-in from the players up front, of course. Good luck with it!