AbstractProtein S‐glutathionylation is one of the important cysteine oxidation events that regulate various redox‐mediated biological processes. Despite several existing methods, there are few proteomic approaches to identify and quantify specific cysteine residues susceptible to S‐glutathionylation. We previously developed a clickable glutathione approach that labels intracellular glutathione with azido‐Ala by using a mutant form of glutathione synthetase. In this study, we developed a quantification strategy with clickable glutathione by using isotopically labeled heavy and light derivatives of azido‐Ala, which provides the relative quantification of glutathionylated peptides in mass spectrometry‐based proteomic analysis. We applied isotopically labeled clickable glutathione to HL‐1 cardiomyocytes, quantifying relative levels of 1398 glutathionylated peptides upon addition of hydrogen peroxide. Importantly, we highlight elevated levels of glutathionylation on sarcomere‐associated muscle proteins while validating glutathionylation of two structural proteins, α‐actinin and desmin. Our report provides a chemical proteomic strategy to quantify specific glutathionylated cysteines.