Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of medical and scientific applications ranging from drug targeting to immunohistochemistry. To maximize the application of this technology in mammalian systems, we recently demonstrated the ability to metabolically biotinylate tagged proteins in mammalian cells using the endogenous biotin ligase enzymes of the mammalian cell. This technology allows site-specific biotinylation without any exogenous reagents and eliminates possible inactivation of the protein of interest by nonspecific biotinylation. Here, we report further expansion of the mammalian metabolic biotinylation technology to enable biotinylation of proteins secreted from mammalian cells and expressed on their cell surface by cosecretion with BirA, the biotin ligase of E. coli. This technique can be used to biotinylate secreted proteins for purification or targeting and also for biotinylating the surfaces of mammalian cells to facilitate their labeling and purification from other nontagged cells.