Updated project metadata. ZDHHC S-acyltransferases catalyze the transfer of fatty acyl groups, primarily palmitate, to cysteine residues of substrate proteins in all eukaryotes. The 23 human ZDHHCs are known to S-acylate an extensive network of proteins essential to normal physiology, including many which are dysregulated in disease. Current technologies for whole proteome analyses of S-acylation do not directly associate a specific ZDHHC with its substrates, making it difficult to link a ZDHHC to specific mechanisms or disease phenotypes. Here, we present a solution to this challenge through development and validation of the first ZDHHC chemical genetic systems. Intact cells expressing a ZDHHC engineered to carry a ‘hole’ mutation in the lipid binding domain were metabolically labeled with a synthetic, chemically-tagged fatty acid probe carrying a matching ‘bump’, resulting in selective fatty acid probe transfer to substrates of the mutant ZDHHC. ZDHHC/probe pairs were exemplified for efficient and selective transfer to ZDHHCs 3, 7, 11, 15 and 20, and adapted to chemical proteomic analysis for ZDHHCs 7, 15 and 20, generating the first de novo substrate profiles for these ZDHHC isozymes across three different cell lines. These data reveal extensive ZDHHC-specific substrate sets across more than 300 total identified substrates, in addition to substrates common between cell lines and between ZDHHCs, and several functionally diverse ZDHHC20 substrates were validated in targeted assays and the sites of S-acylation determined. The chemical genetic platform described here offers a novel and versatile approach to explore ZDHHC substrates and S-acylation biology, with the potential to encompass ZDHHCs across a wide range of eukaryotic organisms. This submission contains data relating to the ZDHHC chemical genetic experiments.