A chemical-genetic system has been developed to map the specific S-acylation substrates of individual ZDHHC enzymes at the whole-proteome level in intact cells. This system uses structure-guided engineering of ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes to enable selective transfer of probes to specific protein substrates. The approach was applied to five human ZDHHCs (3, 7, 11, 15, and 20), identifying over 300 substrates and S-acylation sites across multiple cell lines. The system enables the identification of ZDHHC-specific substrates and S-acylation sites with high selectivity, even in the presence of other ZDHHCs. The chemical-genetic system was validated using a variety of techniques, including chemical proteomics, and demonstrated the ability to identify new S-acylation sites. The system also showed the potential to identify substrates in different cell types and tissues, highlighting the versatility of the approach. The study suggests that this chemical-genetic system could be used to systematically investigate ZDHHC biology and identify potential therapeutic targets. The system also offers a unique approach for resolving ZDHHC isoform-dependent S-acylation at the level of specific PTM sites, while limiting or eliminating probe distribution into non-ZDHHC-dependent pathways. The study highlights the importance of ZDHHCs in health and disease, and suggests that chemical genetics could be used to validate drug targets and discover biomarkers in ZDHHC-associated disease models. The system also has the potential to be adapted for use in a wide range of organisms, including parasites, plants, and fungi. The study demonstrates the potential of chemical genetics to provide a powerful tool for understanding the complex network of S-acylation biology.A chemical-genetic system has been developed to map the specific S-acylation substrates of individual ZDHHC enzymes at the whole-proteome level in intact cells. This system uses structure-guided engineering of ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes to enable selective transfer of probes to specific protein substrates. The approach was applied to five human ZDHHCs (3, 7, 11, 15, and 20), identifying over 300 substrates and S-acylation sites across multiple cell lines. The system enables the identification of ZDHHC-specific substrates and S-acylation sites with high selectivity, even in the presence of other ZDHHCs. The chemical-genetic system was validated using a variety of techniques, including chemical proteomics, and demonstrated the ability to identify new S-acylation sites. The system also showed the potential to identify substrates in different cell types and tissues, highlighting the versatility of the approach. The study suggests that this chemical-genetic system could be used to systematically investigate ZDHHC biology and identify potential therapeutic targets. The system also offers a unique approach for resolving ZDHHC isoform-dependent S-acylation at the level of specific PTM sites, while limiting or eliminating probe distribution into non-ZDHHC-dependent pathways. The study highlights the importance of ZDHHCs in health and disease, and suggests that chemical genetics could be used to validate drug targets and discover biomarkers in ZDHHC-associated disease models. The system also has the potential to be adapted for use in a wide range of organisms, including parasites, plants, and fungi. The study demonstrates the potential of chemical genetics to provide a powerful tool for understanding the complex network of S-acylation biology.