Updated project metadata. The ~160 small GTPases that comprise the Ras superfamily include many major regulators of growth, membrane traffic and the cytoskeleton, and a wide range of diseases are caused by mutations in particular members. They function as switchable landmarks with the active GTP-bound form recruiting to the membrane a specific set of effector proteins. The location and level of the GTP-bound form is precisely controlled by upstream regulators that promote acquisition of GTP (GEFs) or its hydrolysis to GDP (GAPs). We report here MitoID, a method for identifying effectors and regulators by performing in vivo proximity biotinylation with mitochondrial-localized forms of the GTPases. Applying this to 11 human Rab GTPases identified many known effectors and GAPs, as well as putative novel interactors, with examples of the latter validated for Rab2, Rab5 and Rab9. We also show that MitoID can efficiently identify effectors and GAPs of members of other GTPase families such as Cdc42, RhoA, Rac1, Rheb, RalB and N-Ras, and can also identify GEFs by use of GDP-bound forms.