This protocol provides a detailed description of how to design and perform BONCAT experiments using two different bioorthogonal amino acids, L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG), which are both surrogates of L-methionine. It illustrates how incorporation of these noncanonical amino acids into new proteins can be detected in whole cells via copper-catalyzed or strain-promoted azide-alkyne click chemistry and outlines how the visualization of translational activity can be combined with the taxonomic identification of cells via FISH. Last, the protocol discusses potential problems that might be encountered during BONCAT experiments with environmental samples and how they can be overcome.Book chapter describing this BONCAT protocol is published in: Detection of Protein-Synthesizing Microorganisms in the Environment via Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Authors: Roland Hatzenpichler and Victoria J. OrphanT.J. McGenity et al. (eds.), Hydrocarbon and Lipid Microbiology Protocols, Springer Protocols Handbooks, DOI 10.1007/8623_2015_61, © Springer-Verlag Berlin Heidelberg 2015