Quantitative proteome profiling of microorganisms by isotopic labeling of amino acids is still a challenge, because only microorganisms with auxotrophic character are able to embed amino acids into their biomass in a quantitatively correct manner. Here, we describe an isotopic labeling technique (sulfur stable isotope labeling of amino acids for quantification, SULAQ) for the sulfur-containing amino acids cysteine and methionine in a broad range of organisms. The metabolic labeling approach is suitable for gel-based and gel-free protein analysis.