S-palmitoylation attracts more and more attention of neuroscientists, since it is the most common and the only reversible posttranslational lipidation of neuronal proteins. The reversibility of this modification allows it to regulate function of proteins in a dynamic way by controlling their membrane binding, lipid raft localization, trafficking, stability and interactions with other proteins. Selection of the adequate S-palmitoylation analysis method is particularly important for quantification of S-acylation turnover dynamics. The conventional technique is the metabolic labeling with radioactive 3H- or 125I- palmitate and subsequent detection by fluorography. Despite it is widespread, this approach is hazard and time-consuming. The last decade was marked by the real breakthrough in S-palmitoylation field due to the development of new methods, as acyl-biotin exchange (ABE) and metabolic labeling with a palmitic acid analog followed by click chemistry (MLCC). MLCC that is based on metabolic labeling with bioorthogonal palmitic acid analog, linked post-vivo to the specific tag by click chemistry, is an alternative to radiolabeling. Yet this method was used mainly for global qualitative profiling of S-palmitoylated proteins (palmitome), while quantitative palmitoylation analysis of single protein is usually assessed by radiolabeling. Here, we describe the optimization of methodology applicable for quantification of protein palmitoylation based on MLCC, focusing on palmitoylation of the neural cell adhesion molecule (NCAM) by its acyltransferase DHHC3 and the autopalmitoylation of DHHC3. Briefly, quantification is achieved by the enrichment of palmitoylated proteins, linked to biotin tag during MLCC, by streptavidin-sepharose beads, followed by detection of protein of interest (NCAM or DHHC3) using immunoblotting and normalization to total protein level recovered after MLCC. The procedure is relatively safe and fast, with a possibility to access dynamical changes. The key feature of the method is standardization of the palmitoylation assessment of the chosen protein. It increases the reproducibility of observed palmitoylation changes, minimizing the variations introduced by numerous steps of MLCC reaction and protein enrichment. The optimized protocol allows to detect palmitoylation of several proteins in parallel, one of which could serve as an additional internal control.