BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species.