Updated project metadata. Cysteine oxidative modification of cellular proteins is crucial for many aspects of cardiac hypertrophy development. However, integrated dissection of multiple types of cysteine oxidation proteome in cardiac hypertrophy is currently missing. Here we developed a novel discovery platform encompassing a customized biotin switch-based quantitative proteomics pipeline and an advanced analytic workflow to comprehensively profile the landscape of cysteine oxidation in ISO-induced cardiac hypertrophy mouse model. Specifically, we identified a total of 3,717 proteins containing 6,837 oxidized cysteine sites by at least one of reversible cysteine oxidation, cysteine sulfinylation (CysSO2H), and cysteine sulfonylation (CysSO3H). Analyzing the hypertrophy signatures that are reproducibly discovered from computational workflow highlighted a group of fatty acid beta-oxidation enzymes with a continual decreased temporal pattern and a significant decreased abundance in reversible oxidation with no temporal or abundance change in total cysteine, revealing the oxidative regulatory map of fatty acid metabolism in cardiac hypertrophy, which is featured by an overall reduced oxidative metabolism. Our cysteine oxidation platform depicts a dynamic and integrated landscape of the cysteine oxidative proteome, extracted molecular signature, and provided mechanistic insights in cardiac hypertrophy.