Sulfation and phosphorylation are post-translational modifications (PTMs) imparting an isobaric 80 Da addition on the side chain of serine, threonine or tyrosine residues. These two PTMs are often difficult to distinguish due to their similar mass spectrometry (MS) fragmentation patterns. Targeted MS identification of these modifications in specific proteins commonly relies on their prior separation using gel electrophoresis and silver staining. In the present investigation, we report a potential pitfall in the interpretation of these modifications from silver-stained gels due to artifactual sulfation of serine, threonine and tyrosine residues by sodium thiosulfate, a commonly used reagent that catalyses the formation of metallic silver deposits onto proteins. Detailed MS analyses of gel-separated protein standards and E. coli cell extracts indicated that several serine, threonine and tyrosine residues were sulfated using silver staining protocols, but not following Coomassie blue stain. Sodium thiosulfate was identified as the reagent leading to this unexpected side reaction and the degree of sulfation was correlated with increasing concentrations of thiosulfate up to 0.02%, which is typically used for silver staining. The significance of this artifact is discussed in the broader context of sulfation and phosphorylation site identification from in vivo and in vitro experiments.