This article presents a chemical–genetic system to map the protein substrates of specific ZDHHC (zinc finger, DHHC, and Cys) S-acyltransferases at the whole-proteome level in intact cells. The system involves structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes, enabling selective probe transfer to specific protein substrates with high selectivity over wild-type ZDHHCs. The technology was demonstrated for five human ZDHHCs (3, 7, 11, 15, and 20) and applied to generate de novo ZDHHC substrate profiles, identifying over 300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. The platform is expected to elucidate S-acylation biology for a wide range of models and organisms, offering a new approach for systematic investigation of ZDHHC biology and potential therapeutic validation or biomarker discovery.This article presents a chemical–genetic system to map the protein substrates of specific ZDHHC (zinc finger, DHHC, and Cys) S-acyltransferases at the whole-proteome level in intact cells. The system involves structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes, enabling selective probe transfer to specific protein substrates with high selectivity over wild-type ZDHHCs. The technology was demonstrated for five human ZDHHCs (3, 7, 11, 15, and 20) and applied to generate de novo ZDHHC substrate profiles, identifying over 300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. The platform is expected to elucidate S-acylation biology for a wide range of models and organisms, offering a new approach for systematic investigation of ZDHHC biology and potential therapeutic validation or biomarker discovery.