S-fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human disease. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterization of endogenously attached fatty acids is still challenging. here, we optimized and compared two methods widely employed to identify S-fatty-acylated proteins by MS-based proteomics. While these methods identified an overlapping list of fatty-acylated proteins, only alk-16 chemical reporter combined with NH2OH specifically and robustly identified S-fatty-acylated proteins. Alk-16 chemical reporter labeling alone gave a list of S/N/O-fatty acylated proteins, while acyl-RAC provided a list of S-acylated proteins. Acyl-RAC identifies any proteins with a thioester modification and is often misused in the literature to validate S-fatty-acylated proteins. It would be preferable, when validating new S-fatty-acylated proteins, to use acyl-RAC and alk-16 (+/-NH2OH) in combination.