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Protein S-fatty-acylation, the covalent addition of a long-chain fatty acid, predominantly palmitate (S-palmitoylation), to cysteine, is a highly dynamic and regulated process that controls protein function and localization of membrane-associated proteins in eukaryotes. The analysis of S-fatty acylated peptides by mass spectrometry remains challenging due to the hydrophobic and potentially labile thioester linkage of the S-fatty acylated peptides.Here we describe an optimized protocol for the global analysis of S-palmitoylated proteins based on the combination of an alkyne-tagged chemical reporter of palmitoylation, alk-16 with hydroxylamine-selective hydrolysis of thioester bonds. This protocol decreased the number of false positive proteins and was applied to identify S-fatty acylation sites, providing modification sites for 44 proteins out of the 106 S-fatty acylated proteins identified.