A new biotin ligase, TurboID, has been developed that enables efficient proximity labeling in living cells and organisms. This enzyme, along with its smaller variant miniTurbo, outperforms existing biotin ligases like BioID and BioID2 in terms of catalytic efficiency, allowing for rapid labeling (within 10 minutes) without the need for long incubation times or toxic chemicals. TurboID and miniTurbo can label proteins within a few nanometers of the enzyme, enabling the identification of protein interactions and subcellular compartments. The enzymes were engineered through yeast display-based directed evolution, which involved mutagenesis, selection, and amplification of variants with improved activity. TurboID is the most active of the two, but miniTurbo offers advantages in terms of temporal control and reduced background labeling. The enzymes have been tested in a variety of organisms, including yeast, flies, and worms, demonstrating their broad applicability. TurboID and miniTurbo also show improved performance in different cellular compartments, such as the nucleus, mitochondria, and endoplasmic reticulum. The new enzymes offer a more efficient and less toxic alternative to existing methods for studying protein interactions and subcellular localization. The study highlights the potential of TurboID and miniTurbo for use in proteomic analysis and in vivo applications.A new biotin ligase, TurboID, has been developed that enables efficient proximity labeling in living cells and organisms. This enzyme, along with its smaller variant miniTurbo, outperforms existing biotin ligases like BioID and BioID2 in terms of catalytic efficiency, allowing for rapid labeling (within 10 minutes) without the need for long incubation times or toxic chemicals. TurboID and miniTurbo can label proteins within a few nanometers of the enzyme, enabling the identification of protein interactions and subcellular compartments. The enzymes were engineered through yeast display-based directed evolution, which involved mutagenesis, selection, and amplification of variants with improved activity. TurboID is the most active of the two, but miniTurbo offers advantages in terms of temporal control and reduced background labeling. The enzymes have been tested in a variety of organisms, including yeast, flies, and worms, demonstrating their broad applicability. TurboID and miniTurbo also show improved performance in different cellular compartments, such as the nucleus, mitochondria, and endoplasmic reticulum. The new enzymes offer a more efficient and less toxic alternative to existing methods for studying protein interactions and subcellular localization. The study highlights the potential of TurboID and miniTurbo for use in proteomic analysis and in vivo applications.