Arkham Origins Incident
During Batman's fight with Deathstroke on the Final Offer, if the player non-canonically failed, one game-over screen ended with Deathstroke unholestering his handgun (which appeared to be a Desert Eagle) and executing Batman with it. Deathstroke also used a laser sighted pistol that shot sniper-like shots if the player put a large amount of distance between him and Batman.
The Joker used one to execute two of his own henchmen, and proceeded to point it toward his own head, only to be stopped by Batman. Later in the game, Joker also planned on shooting Warden Joseph with the gun, but Captain Gordon blocked the bullet with his bulletproof vest.
Arkham Origins Multiplayer
While handguns weren't usable by Elite gang members, Joker again used his Ace of Spades and his King of Hearts. While the former shot a single instantly-killing bullet, the latter shot multiple explosive bullets in a rapid succession.
Arkham Origins Blackgate Incident
Black Mask used one to execute an technician. Black Mask also used two handguns during his boss fight with Batman. The guns in his boss fight however, had a very fast rate of fire, and were capable of killing Batman in mere seconds.
Arkham Asylum Incident
The handgun had one in-game appearance during the third Scarecrow Nightmare. It was used by the Joker in a non-combat situation. He used it to shoot a restrained and "insane" Batman in the head.
Arkham City Incident
The Handgun returned in Arkham City, but was used in a combat situation by Two-Face. It did medium damage. It made another appearance in a cutscene, and was wielded by the Joker. It was the least used firearm.
Arkham Knight Incident
Handguns make several appearances through the game, but weren't wielded by enemies. Several guns that belonged to super-villains could be seen at the GCPD Lockup Evidence Room. During the time Joker briefly took over Batman on Stagg's Airships, he hallucinated executing Scarecrow with a revolver. Soon afterwards, under the influence of Scarecrow's Fear Toxin, Batman believed that he saw Oracle commit suicide with one. Henry Adams murdered the other three mutated Jokers using one, and later shot himself. Scarecrow used one to threaten both Robin and Batman near the end of the game.
Jason Todd dual wielded two modified handguns, which could be merged to create a sniper rifle. He also uses the same guns in the Red Hood Story Pack DLC, but their rifle form wasn't usable by the player.
If the player had a PS3, the Play as the Joker DLC, allowed the player to control Joker and use his handgun in combat and predator challenges. It was always an instant kill and acted like Batman's Batarangs, as it was a projectile that could be aimed manually or could quickly be unholstered and shot/thrown by the tap of a button. However, the player could only fire the firearm once a round, which meant that the Joker seemed to only carry a single bullet with him. The bullet could, however, travel through multiple guards.
While playing as Red Hood, the player could freely use his dual handguns. As his primary quickfire gadget, they could be used to stop charging enemies. Unless the enemy was using a Riot Shield or was bulletproof, they were always an instant kill, and could be used for an unlimited amount of times. They were also utilized in finishers.
There were a few drawbacks however, as Jason Todd shot multiple rounds to the targeted enemy, and was vulnerable to other enemies while doing so. They also produced a lot of noise, so using them in Predator Mode could lead to detection. When aimed, they always went into first person view.
Combining them with the Flashbang, allowed Jason to perform an unique version of a Beatdown. After throwing a Flashbang, if the enemy was far enough, each press of the attack button shot a round at the enemy.
Joker's Ace of Spades can be found in the GCPD Evidence Room along with the rest of Joker's confiscated gear.