S-acylation is the covalent addition of a fatty acid, most generally palmitate onto cysteine residues of proteins through a labile thioester linkage. The death receptor CD95 is Spalmitoylated and this post-translational modification plays a crucial role on CD95 organization in cellular membrane and thus on CD95-mediated signaling. Here, we describe the non-radioactive detection of CD95 S-acylation by acyl-biotin exchange chemistry in which a biotin is substituted for the CD95-linked fatty acid. This sensitive technique, which depends on the ability of hydroxylamine to specifically cleave the thioester linkage between fatty acids and proteins, relies on 3 chemical steps: (i) blockage of free thiols of non-modified cysteine residues, (ii) hydroxylamine-mediated cleavage of thioester-linked fatty-acids to restore free thiols and (iii) biotinylation of free thiols with a thiol reactive biotinylation agent. Resulting biotinylated proteins can be easily purified by an avidin capture and analysed by SDS-PAGE and immunoblotting.