Reversible protein S-acylation dynamically regulates the subcellular localization, activity, and stability of proteins, and plays a pivotal role in various diseases such as cancer and neurodegenerative disorders. Nevertheless, a system-wide analysis of S-acylation in human pathophysiology is still elusive, largely due to the lack of a sensitive method for proteome-wide characterization of S-acylation. Via systematic optimization of our original Palmitoyl-protein Identification and Site Characterization (PalmPISC) method, we established the next-generation PalmPISC (ngPalmPISC). The application of ngPalmPISC to human LNCaP cells identified 1,682 known and 1,175 novel candidate S-acylated proteins as well as 1,648 candidate S-acylation sites—a coverage of an order of magnitude larger than any previous studies. Moreover, the large set of S-acylation sites allowed motif enrichment analysis, resulting in the identification of two novel S-acylation motifs CxL and FxC (p<0.01). The ngPalmPISC method and the comprehensive human S-acylproteome dataset are expected to facilitate our system-wide understanding of protein S-acylation in pathophysiology and the discovery of novel biomarkers and therapeutic targets.