In eukaryotic cells, specific mechanisms allow selective packaging of proteins and lipids into transport vesicles, which can then specifically recognize the membrane of the acceptor compartment and fuse with it to deliver their cargo. Formation, transport and docking of vesicles are based on a complex network of interactions between regulatory molecules and structural components. Small GTP-binding proteins have emerged as master regulators of all steps of vesicle trafficking. In this chapter, we will first present the general mechanisms of GTP-binding protein function that are based on their ability to bind to and hydrolyze GTP. Specific methods commonly used to study GTP-binding protein activation will be briefly de- scribed. The last section will then review, through selected examples, the different ways by which proteins belonging to the different families of small GTP-binding proteins control various aspects of intracellular vesicle trafficking.