Post-translational modifications (PTM) represent the largest source of proteome diversity, regulating the function and fate of almost every eukaryotic protein. One of these is S-acylation, which refers to the post-translational covalent attachment of long-chain fatty acids onto thiol groups in surface-exposed cysteine residues. The combination of two enzyme classes, S-acyltransferases and thioesterases dynamically control the levels of this PTM in cellular proteins. The former, includes a family of 23 isoenzymes commonly known as ZDHHCs due to their conserved catalytic motif, which together S-acylate every eukaryotic protein. S-acylation has been found to participate in numerous cellular functions including protein localisation, trafficking, signalling or gene expression and consequently, misregulation of S-acylation has been associated to neurodegenerative diseases and cancer. To date, the tool set available to study the S-acylated proteome remains scarce and without small molecule modulators of individual ZDHHCs, their biological significance cannot be selectively detangled from the rest of the family. Here, I present the design and development of a novel chemical genetic approach to unravel substrate scope of ZDHHC20 via chemical proteomics. For this, ZDHHC20 was engineered to contain a mutation in the lipid binding domain, which allowed both the selective binding of a lipid probe and its transfer to substrates. This allowed the curation of the first substrate list for ZDHHC20 in HEK293T cells, several of which were validated in targeted assays. Their analysis depicted a strong involvement of ZDHHC20 in vesicular trafficking from the endoplasmic reticulum and Golgi apparatus. This relationship was further explored using ER-localised TurboID to compare ER-resident and secreted proteins in ZDHHC20 KO and wildtype backgrounds. These tools represent a novel and versatile perspective from which to explore the substrate scope of individual ZDHHC enzymes and their biological relevance in health and diseased cellular states.