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The primary goal of the game is to complete nine assassinations. To achieve this goal, the player must use stealth and a variety of intelligence gathering tactics to collect information on their target. These tactics include eavesdropping, forceful interrogation, pickpocketing, and completing tasks for Informers (other assassins who will give you information in exchange for assassinating targets or collecting flags). Additionally, the player may take part in any number of side missions, including climbing tall towers to map out the city and saving citizens that are being threatened or harassed by the city guards. There are also various side quests unrelated to the story such as hunting down and killing Templars and flag collecting.

The game takes place primarily in a kingdom consisting of four main cities: Jerusalem, Acre, Damascus and Masyaf, the assassins' stronghold; all but Masyaf contain three sections that are unlocked over the course of the game. An approximation of the land lying between these areas is present in the game as "The Kingdom." Each city features a highly detailed environment populated by many people of different types, and as the player interacts with his/her environment, the people around Altaïr respond in logical and predictable ways. For example, Altaïr can climb buildings, causing people to gather around and comment on his unusual behavior. This in turn can attract the unwanted attention of nearby guards, who may then choose to attack him.

The player is made aware of how noticeable Altaïr is to enemy guards as well as the current state of alert in the local area. To perform many of the assassinations and other tasks, the player must consider the use of both "low profile" and "high profile" commands. "Low profile" commands allow Altaïr to blend into nearby crowds, gently pass by other citizens, or other non-threatening tasks that can be used to hide and reduce the alertness level. "High profile" commands are more noticeable, and including running, scaling the sides of buildings to climb to higher vantage points, and attacking foes; performing these actions at certain times may raise the local area's awareness level. Once the area is at high alert, the player must use both low profile commands as well as aiming to climb away from ground level or using hiding spaces to reduce the alertness level. The player, as they complete the assigned assassinations, will gain either new weapons or new combat skills including counter-attacks and attack dodges. Besides his fists, Altaïr's weaponry includes an assassin's sword for standard combat, a short blade, and throwing knives which are used in conjunction with the short blade. Altaïr also gains the use of a hidden retractable blade on his left arm that can be used for killing targets at very close quarters without creating any immediate alerts (although, if others come across the body, the alertness level may be raised). This is the most noticeable difference between low and high profile. A low profile assassination can be performed within as little as a few feet of other guards or soldiers without attracting attention, whereas a high profile assassination attack will be noticed by everyone within eyesight.

The player's health is described as the level of synchronization between Desmond and Altaïr's memories; should Altaïr take damage, some amount of synchronization is lost, and if all synchronization is lost, the current memory that Desmond is experiencing will be restarted at the last checkpoint. When the synchronization bar is full, the player has the additional option to use "eagle vision" which causes the computer-rendered memory to highlight all visible characters in colors corresponding to whether they are friend or foe or even the target of their assassination. Due to Altaïr's memories being rendered by the computer of the Animus project, the player may experience "glitches" in the rendering of the historical world, which may help the player to identify targets, or can be used to alter the viewpoint during in-game scripted scenes should the player react fast enough when they appear.