Redox post-translational modifications (PTM) are emerging as important regulatory mechanisms in signaling and pathogenesis. Cysteine (Cys) is the most redox active amino acid and is a target for these PTM, some of which are biologically reversible (e.g. disulfides, sulfenic acid) while others (sulfinic [Cys-SO₂H] and sulfonic [Cys-SO₃H] acids) are considered irreversible. We have developed enrichment methods to examine these PTM on a proteome-wide scale. Rapid and specific alkylation of free Cys, followed by thiol-based reduction and resin capture by thiol-disulfide exchange chemistry was applied to isolate reversibly modified Cys-containing peptides. The method was applied to a complex protein lysate generated from rat myocardial tissue and 6559 unique Cys-containing peptides from 2694 proteins were identified by tandem mass spectrometry (MS/MS). We next developed an enrichment method to isolate Cys-SO₂H/SO₃H-containing peptides from complex tissue lysates. The method is based on electrostatic repulsion of Cys-SO₂H/SO₃H-containing peptides from cationic resins (i.e. ‘negative’ selection) followed by ‘positive’ selection using hydrophilic interaction liquid chromatography (HILIC). We identified 181 Cys-SO₂H/SO₃H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H₂O₂ (<100 µM) or to ischemia / reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO₂H/SO₃H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair. Finally, we have combined these methods to enable multiplexed quantitative analysis of reversible/irreversible Cys redox PTM in response to I/R and in the presence of a broad-spectrum antioxidant (N-2-mercaptopropionyl glycine, MPG). We quantified >1350 Cys sites that are reversibly and/or irreversibly oxidized by I/R, including many sites that are protected by MPG. This technique allows for the quantitative profiling of reversible/irreversible Cys PTMs in response to oxidant / antioxidant stimulus, and their delineation within the context of protein abundance, during I/R injury and cardioprotection.