Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform comparative analysis in cell biology.