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The Hunter ranger can choose the Colossus Slayer option for their 3rd-level Hunter's Prey feature.

Suppose that on my turn, my Hunter ranger hits my opponent and gets to use Colossus Slayer. Then on the enemy's turn, they turn around and run away, giving me an opportunity attack. My opportunity attack hits. As the hit is occurring on the enemy's turn, does that mean I can use Colossus Slayer twice in a round, once on my turn and once on the enemy's turn?

Colossus Slayer states that its damage is "only once per turn". As Colossus Slayer does not explicitly state that it happens on "your turn", I can see some room for confusion or debate over this.

Does the Hunter ranger's Colossus Slayer feature work with an opportunity attack?

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Yes!

As you've noted, Colossus Slayer limits to only once per turn. If the opportunity attack occurs off your turn, then you can use it again because you are not using it 'on your turn.'

Do note that you still only get one Reaction, so you effectively you can really just do this twice per round (once on your turn, once off your turn with a reaction spent.)

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To add to NautArch's answer, the answer to your question is yes - Colossus Slayer does work with opportunity attacks - but there are some interesting edge cases where you can do the extra damage from Colossus Slayer three (or more) times in a round.

So the problem is that Colossus Slayer is limited per turn. So if you attack and hit once in your turn you can't any more activation in that turn (short of wish shenanigans). As per the correct answer you can get a second activation during another characters turn via attack of opportunity (or via a Battle Master fighter's Commander's Strike maneuver). But an extra 2d8 per round doesn't really feel like it's going to kill a colossus, does it?

The easiest and stupidest way to get more damage out of Colossus Slayer is to be a multiclassed level 17 Wizard / level 3 Ranger and use time stop to get multiple turns in one round. Unfortunately, if you have a level 20 character and do this, then the god of action economy will appear in real life and smite you.

But there is an Unearthed Arcana fighting style called Tunnel Fighter (from UA: Light, Dark, Underdark!), which can trigger the extra damage from Colossus Slayer many, many more times in a single round without incurring divine intervention (emphasis mine):

You excel at defending narrow passages, doorways, and other tight spaces. As a bonus action, you can enter a defensive stance that lasts until the start of your next turn. While in your defensive stance, you can make opportunity attacks without using your reaction, and you can use your reaction to make a melee attack against a creature that moves more than 5 feet while within your reach.

With this, you can get more than two Colossus Slayer strikes per round if you can get multiple enemies to provoke opportunity attacks. Great. Now all we need is a bunch of enemies to provoke opportunity attacks from our ranger.

Strictly speaking, depending on party size, campaign level, preparation and DM willingness you could potentially have vast arrays of enemies leaving your reach and provoking opportunity attacks in one round via feats, spells, class abilities, glyphs of warding, and many, many other exploits. Ultimately, you could force dozens of enemies into the range of our Tunnel Fighter ranger to get dozens of extra 1d8 damage.

Why? Because.

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    \$\begingroup\$ Once you attack while in time stop, the stop ends. Additionally, commanders strike also takes your reaction, so you can't do an OA and the strike. \$\endgroup\$ – NautArch May 1 at 18:37
  • \$\begingroup\$ You're right that time stop ends if you effect another creature but I believe it should be possible for a lvl 17 wiz to get at least 3 Colossus strikes in a single combat round unaided (without using glyphs of warding or magical items) by combining time stop, haste and other spells, the idea being ending TS while attacking and then forcing OT by casting fear or another spell. Regarding commanders strike I just brought it up as alternative to OA as a way to trigger a CS since it also uses a reaction \$\endgroup\$ – Nicholas McKay May 1 at 19:08
  • \$\begingroup\$ heh, I'd forgotten about Tunnel Fighter cheese. Thanks for reminding me. \$\endgroup\$ – KorvinStarmast May 1 at 19:34

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