Assassin signals will be unlocked through standard progression of the story. The red arrow below your health bar represents your Assassin Signal. This arrow shows how many assassin signals you have available, and how much time remaining before your assassins return to signaling range.
In order to use your Assassins Signal, follow the guidelines below:
1. The arrow below the health bar needs to be filled red
2. Ezio needs to be within range of the target
3. Your target needs to be flashing
4. Press LB(Xbox 360) or L1(PS3) to send assassins out and after a target