Rab GTPases are the largest family of small GTPases, with 66 members in humans, and play essential roles in membrane trafficking by recruiting effector proteins to the correct membrane at the correct time. Indeed, many proteins involved in membrane trafficking form direct interactions with Rab GTPases as either Rab effectors, or Rab activators and exchange factors; thus, identifying proteins which interact with Rabs can provide useful insights to the mechanisms underlying protein trafficking processes. In this work, we have combined in silico analyses using AlphaFold2 (AF2) with biophysical and cellular methods to systematically identify and characterise Rab interacting partners. Initially, we used AF2 modelling to study the interaction between the tumour-suppressor protein Nischarin and Rab14. Using a point mutation we have validated the binding site predicted by AF2, and have found that overexpression of this complex in mammalian cells causes aggregation of vesicular structures. Given that AF2 was successfully able to model the Nischarin-Rab14 interaction, we then applied AF2 modelling to triage published proteomics datasets to identify novel interactors of Rab GTPases. For the 20 Rabs tested thus far, AlphaFold was able to model 121 interactions with high confidence, including both known interactions and those that are unreported in the literature. Notably, our modelling has suggested that several syntaxin family proteins (which act as SNAREs in membrane fusion) interact with Rab GTPases. Two of these interactions predicted by AlphaFold—the Syntaxin1A-Rab6B interaction and the Syntaxin7-Rab14 interaction—have been validated using in vitro methods. Furthermore, we have found that the BEACH domain—a highly conserved domain of unknown function—interacts with Rab GTPases. Surprisingly, both our AlphaFold2 models and in vitro assays suggest that the BEACH domain is functions as a GTPase activating protein (GAP), rather than an effector. Overall, these findings demonstrate that using AlphaFold2 modelling in combination with in vitro methods can accelerate the identification and characterisation of protein-protein interactions.