Secretion and endocytosis are essential processes in eukaryotic cells, executed and tightly regulated by the cell’s endomembrane system. It is widely accepted that small GTPases of the Ras superfamily are major regulators of membrane trafficking and signalling, ensuring specificity and efficiency through their spatiotemporal regulation.Identifying the proteins interacting with small GTPases is vital for understanding how the small G proteins are regulated and where they function. With the aim of eliminating certain limitations posed by many traditional methods of studying protein-protein interactions, an adaptation of the recently developed in vivo proximity labelling technique BioID was used in this study. Here, BioID was redirected to the ectopic location of the surface of mitochondria (hereafter called MitoID) with the aim of restricting the non-specific background. Applying this method to 25 small GTPases of the Ras superfamily in their active and inactive states allowed for a direct and clean comparison between the putative interactomes, identifying both known effectors and regulators as well as putative novel interactors for most tested GTPases. Several potential novel interactions were validated through GST affinity chromatography and/or microscopy, namely with the Rab GTPases Rab2A (ARFGEF3/BIG3, STAMBPL1), Rab5A (OSBPL9, TBCK), Rab9A (HPS3, NDE1), and Rab11A/B (ALS2). Furthermore, numerous novel interactors were identified for Rab1A and Rab1B, two mammalian paralogues of the yeast protein Ypt1 that are known to be key regulators in ER to Golgi trafficking and suggested to play a role in the regulation of autophagy. The newly identified proteins include Rabaptin5, which is a key player in the regulation of endosomal trafficking, PPP1R37, a protein of unknown function, CALCOCO1 and CLEC16A, which are potentially involved in autophagy, and several components of the phosphatidylinsositol-3-phospate kinase (PI3K) complex I, which is known to be a major autophagic regulator. In collaboration with the Williams group, the PI3K complex I was shown to be a bona fide Rab1A regulator. Furthermore, direct binding assays showed that CALCOCO1, CLEC16A, and PPP1R37 all bind to Rab1A and Rab1B independently of adaptor proteins or other factors. More specifically, the Rab1 binding site on PPP1R37 was shown to be located on the C-terminal and central regions that contain multiple leucine rich repeats. Collectively, this study has shown that MitoID is an effective and powerful tool to study protein-protein interactions, and has identified and confirmed several novel interactors of small GTPases which can aid in gaining understanding of how major processes such as vesicle trafficking and autophagy are regulated.