A mechanism for the cellular effects of the gas H2S, is the oxidative modification of protein cysteine residues. We developed a quantitative proteomics tool to profile protein S-persulfidation in cellular proteomes. We discovered a Redox Thiol Switch of S-glutathioinylation to S- persulfidation of proteins, among them enzymes in cellular energy metabolism. This work allows identification of redox regulation of cysteine residues of proteins in physiological and disease states and can assist design of therapeutics for diseases such cancer and diabetes. Display omitted Highlights •Develop a TMT-based proteomics tool to profile cysteine persulfides in the cellular proteomes.•Discover a Redox Thiol Switch from protein S-glutathioinylation to S-persulfidation (RTSGS) with implications in the regulation of cellular energy metabolism under oxidative stress. The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H2S has been shown to persulfidate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H2S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTSGS). We propose that the RTSGS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.