Oxidatively modified thiol groups of cysteine residues are known to modulate the activity of a growing number of proteins. In this study, we developed a fluorescence‐based thiol modification assay and combined it with two‐dimensional gel electrophoresis and mass spectrometry to monitor the in vivo thiol state of cytoplasmic proteins. For the Gram‐positive model organism Bacillus subtilis our results show that protein thiols of growing cells are mainly present in the reduced state. Only a few proteins were found to be thiol‐modified, e.g. enzymes that include oxidized thiols in their catalytic cycle. To detect proteins that are particularly sensitive to oxidative stress we exposed growing B. subtilis cells to diamide, hydrogen peroxide or to the superoxide generating agent paraquat. Diamide mediated a significant increase of oxidized thiols in a variety of metabolic enzymes, whereas treatment with paraquat affected only a few proteins. Exposure to hydrogen peroxide forced the oxidation especially of proteins with active site cysteines, e.g. of cysteine‐based peroxidases and glutamine amidotransferase‐like proteins. Moreover, high levels of hydrogen peroxide were observed to influence the isoelectric point of proteins of this group indicating the generation of irreversibly oxidated thiols. From the overlapping set of oxidatively modified proteins, also enzymes necessary for methionine biosynthesis were identified, e.g. cobalamin‐independent methionine synthase MetE. Growth experiments revealed a methionine limitation after diamide and hydrogen peroxide stress, which suggests a thiol‐oxidation‐dependent inactivation of MetE. Finally, evidence is presented that the antibiotic nitrofurantoin mediates the formation of oxidized thiols in B. subtilis.