Updated project metadata. Stable isotope labeling with amino acids in cell culture (SILAC) is a robust proteomics method with the advantages of reproducibility and easy handling. This method is popular for the analysis of mammalian cells. However, amino acid conversion in bacteria decreases the labeling efficiency and quantification accuracy, limiting the application of SILAC in bacterial proteomics to auxotrophic bacteria or single labeling with lysine. In this study, we found that adding high concentrations of isotope-labeled (heavy) and natural (light) amino acids into SILAC minimal medium can efficiently inhibit the complicated amino acid conversions between each other. This simple and straightforward strategy facilitated the full incorporation of amino acids into the bacterial proteome with good accuracy. The high labeling efficiency can be reached in different bacteria by slightly modifying the supplement of amino acids in culture media, promoting the widespread application of SILAC technique in bacterial proteomics.