The game takes place between the events of the Battle of Forli and Bonfire of the Vanities (the extra content available for Assassin's Creed II) in the year 1491. Ezio Auditore da Firenze is to meet with another assassin, Antonio, the leader of the Thieves Guild in Venice. Upon finding Antonio, Ezio finds that there is a Spaniard that requires his help by the name of Luis Santagel whose accomplice, Christoffa Corombo, had conversed with Rodrigo Borgia who had shown interest in Christoffa's plans to sail West. Luis suspects a trap and requests Ezio's help to rescue his accomplice. It turns out Luis' suspicions were well founded, and Ezio discovered that it was indeed a trap but successfully rescued Christoffa. Through the events of the game, Ezio meets Helene Dufranc who was born into the assassins but is dismissive of their Order. After travelling to Spain to rescue several assassins who were being held by the Spanish Inquisition, Ezio finds out about the plans of the Templar to reach the New World.
The gameplay takes a side-scrolling perspective, and all action is rendered on a 2D plane. Many of the elements from Assassin's Creed II made their way into the gameplay of Discovery, including ledge assassinations, counter-attacks and the awareness system which allows Ezio to spot guards before he is seen.
There are three distinct mission types: Chase, Stealth and Normal missions. Chase missions are tests of your ability to speed through a level often with a timer or a person that has to be reached. The main point of the stealth missions is to stay out of sight through means of hay stacks, barrels, ledges as well as assassinating a portion of the guards.
The guards have been sorted into different sets, and as such, have different defenses and abilities. Archers, brutes and seekers all make an appearance in the game. Unfortunately, Ezio is unable to disarm enemies this time around so he cannot obtain heavy weapons. This means he has to fight enemies head-on or perform a stealth assassination.
Like in the console version of Assassin's Creed II, Discovery has wanted posters that can be ripped down, but instead of reducing Ezio's wanted level it will give him an increased sync. In addition to finding wanted posters, the player can find hidden treasure that contain extra content, such as art work from the console version.