Protein interaction networks and compartmentalization are crucial for signaling and regulatory processes in cells. Enzyme-catalyzed proximity labeling (PL) has emerged as a method to study these interactions in living cells, but current methods require long labeling times or use chemicals with limited cell permeability or high toxicity. The authors engineered two promiscuous mutants of biotin ligase, TurboID and miniTurbo, which enable 10-minute PL in cells with non-toxic and easily deliverable biotin. TurboID extends biotin-based PL to flies and worms. TurboID and miniTurbo show significantly higher activity than BioID or BioID2, with TurboID being the most active. These enzymes produce proteomic datasets of similar quality to BioID labeling for 18 hours, in terms of specificity, coverage, and labeling radius. TurboID and miniTurbo are also more active than BioID in various organelles and in vivo applications, such as in bacteria, yeast, flies, and worms. However, TurboID can sequester endogenous biotin and cause toxicity if expressed ubiquitously, and miniTurbo exhibits less stability and smaller size compared to TurboID.Protein interaction networks and compartmentalization are crucial for signaling and regulatory processes in cells. Enzyme-catalyzed proximity labeling (PL) has emerged as a method to study these interactions in living cells, but current methods require long labeling times or use chemicals with limited cell permeability or high toxicity. The authors engineered two promiscuous mutants of biotin ligase, TurboID and miniTurbo, which enable 10-minute PL in cells with non-toxic and easily deliverable biotin. TurboID extends biotin-based PL to flies and worms. TurboID and miniTurbo show significantly higher activity than BioID or BioID2, with TurboID being the most active. These enzymes produce proteomic datasets of similar quality to BioID labeling for 18 hours, in terms of specificity, coverage, and labeling radius. TurboID and miniTurbo are also more active than BioID in various organelles and in vivo applications, such as in bacteria, yeast, flies, and worms. However, TurboID can sequester endogenous biotin and cause toxicity if expressed ubiquitously, and miniTurbo exhibits less stability and smaller size compared to TurboID.