The organization of intracellular transport processes is adapted specifically to different cell types, developmental stages, and physiologic requirements. Some protein traffic routes are universal to all cells and constitutively active, while other routes are cell-type specific, transient, and induced under particular conditions only. Small GTPases of the Rab (Ras related in brain) subfamily are conserved across eukaryotes and regulate most intracellular transit pathways. The complete sets of Rab proteins have been identified in model organisms, and molecular principles underlying Rab functions have been uncovered. Rabs provide intracellular landmarks that define intracellular transport sequences. Nevertheless, it remains a challenge to systematically map the subcellular distribution of all Rabs and their functional interrelations. This task requires novel tools to precisely describe and manipulate the Rab machinery in vivo. Here we discuss recent findings about Rab roles during development and we consider novel approaches to investigate Rab functions in vivo.