Protein S-glutathionylation is one of the major cysteine oxidations regulating redox signaling and oxidative stress. In this study, we developed a glutathione-derived chemical tool, namely dehydroglutathione, that can introduce a non-reducible glutathionylation mimic to protein, which can be utilized for analyzing the functional effects of glutathionylation. Dehydroglutathione reacts with protein cysteines to form a thio-ether based glutathione modification in proteins. We applied dehydroglutathione to fatty acid binding protein 5 (FABP5). Dehydroglutathione modification in FABP5 was analyzed by LC-MS/MS to identify the cysteine sites of modification.