Cell-selective metabolic labeling of proteins with noncanonical amino acids enables the study of proteomic changes in specified subpopulations of complex multicellular systems. For example, azidonorleucine (Anl) and 2-aminooctynoic acid, both of which are activated by an engineered methionyl-tRNA synthetase (designated NLL-MetRS), are excluded from proteins made in wild-type cells but incorporated readily into proteins made in cells that carry NLL-MetRS. To expand the set of tools available for cell-selective metabolic labeling, we sought a MetRS variant capable of activating propargylglycine (Pra). Pra was chosen as the target amino acid because its alkynyl side chain can be selectively and efficiently conjugated to azide-functionalized fluorescence probes and affinity tags. Directed evolution, using active-site randomization and error-prone PCR, yielded a MetRS variant (designated PraRS) capable of incorporating Pra at near-quantitative levels into proteins made in a Met-auxotrophic strain of Escherichia coli cultured in Met-depleted media. Proteins made in E. coli strains expressing PraRS were labeled with Pra in Met-supplemented media as shown by in-gel fluorescence after conjugation to Cy5-azide. The combined use of NLL-MetRS and PraRS enabled differential, cell-selective labeling of marker proteins derived from two bacterial strains cocultured in media supplemented with Met, Anl, and Pra. Treatment of the mixed marker proteins by sequential strain-promoted and copper(I)-catalyzed cycloadditions allowed straightforward identification of the cellular origin of each protein.