The GTPases of the Ras superfamily regulate cell 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 GTPases are precisely controlled by 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 mitochondrially-localized forms of the GTPases. Applying this to 11 human Rab GTPases identified many known effectors and GAPs, as well as putative novel effectors, with examples of the latter validated for Rab2, Rab5, Rab9 and Rab11. MitoID can also efficiently identify effectors and GAPs of Rho and Ras family GTPases such as Cdc42, RhoA, Rheb, and N-Ras, and can identify GEFs by use of GDP-bound forms.