Cysteine is very susceptible to reactive oxygen species. In response; posttranslational thiol modifications such as reversible disulfide bond formation have arisen as protective mechanisms against undesired in vivo cysteine oxidation. In Gram-negative bacteria a major defense mechanism against cysteine overoxidation is the formation of mixed protein disulfides with low molecular weight thiols such as glutathione and glutathionylspermidine. In this review we discuss some of the mechanistic aspects of glutathionylspermidine in prokaryotes and extend its potential use to eukaryotes in proteomics and biochemical applications through an example with tissue transglutaminase and its S-glutathionylation.