The Ras superfamily consists of over 150 low molecular weight proteins that cycle between an inactive guanosine diphosphate (GDP)-bound state and an active guanosine triphosphate (GTP)-bound state. They are involved in a variety of signal transduction pathways that regulate cell growth, intracellular trafficking, cell migration, and apoptosis. Several methods have been devised to detect and characterize the interacting partners of small GTPases with the aim of better understanding their physiological function in normal cells and tumor cells. The Rab (Ras analog in brain) proteins form the largest family within the Ras superfamily. Rab proteins regulate vesicular trafficking pathways, behaving as membrane-associated molecular switches. The guanine nucleotide-binding status of Rab proteins is modulated by three different classes of regulatory proteins, which have been extensively studied for the Rab molecules but also for other subfamilies of the Ras superfamily. Furthermore, numerous effector molecules have been isolated especially for the Rab subfamily of proteins, which interact via a Rab-binding domain (RBD) and are recruited afterwards to specific sub-cellular compartments by the Rab proteins.