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A Chemical Proteomics Method to Quantify Cysteine S-Acylation S-acylation, often referred to as S-palmitoylation, is a reversible and dynamic posttranslational modification that corresponds to the addition of a long-chain fatty acid to cysteine (Cys) residues. Established mass spectrometry-based chemoproteomics methods have improved our understanding of the S-acylation proteome, notably by identifying hundreds of S-acylated proteins, sometimes with the modified Cys. However, the precise quantification of S-acylation levels for each Cys within a single sample remains challenging at the proteome level. Quantification of S-acylation levels is critical to further our understanding of protein S-acylation in cellular function and its role in health and diseases. We report here the development of an S-acylation quantification workflow based on the sequential labeling of free Cys and S-acylated Cys with isotopic labeling reagents. The workflow was extensively optimized, notably by comparing the number of sites identified with two alkyne-tagged Cys-reactive isotopic probes and four azido-tagged biotin-based capture reagents. By integrating this enhanced workflow with high-field asymmetric waveform ion mobility spectrometry (FAIMS) on LC–MS/MS instruments for the separation of labeled peptides, over 17,000 unique Cys could be quantified in biological samples. Application of the S-acylation quantification workflow to cellular proteomes allowed for the quantification of S-acylation levels in a HeLa proteome. We also identified dynamic S-acylation changes in response to autophagy induction. https://pubs.acs.org/doi/abs/10.1021/acschembio.5c00824