Protein S‐acylation (palmitoylation) reversibly modulates protein localization, and thus regulates protein activity, stability, and complex formation. Accumulating evidence suggests that S‐acylation plays a key role in the development and progression of various diseases, such as cancer and neurodegenerative diseases. Previously, we established an S‐acylproteomics method termed Palmitoyl‐protein Identification and Site Characterization (PalmPISC), and identified 67 known and 331 novel candidate S‐acylated proteins from human prostate cancer DU145 cells (Yang et al., Mol Cell Proteomics, 2010, 9: 54–70). In this study, we developed a next‐generation PalmPISC (ngPalmPISC) method that enables proteome‐wide characterization of S‐acylated proteins. Using ngPalmPISC, we identified 2,063 high‐confidence and 528 medium‐confidence candidate S‐acylated proteins from human prostate cancer LNCaP cells. Of these, 1,174 (56.9%) high‐confidence and 242 (45.8%) medium‐confidence candidates are contained in the SwissPalm database, corroborating the effectiveness of our ngPalmPISC method in identifying S‐acylated proteins. In addition, 1,191 and 457 cysteine residues were identified as candidate S‐acylation sites with high and medium confidence, respectively. To our knowledge, this represents the first proteome‐wide characterization of human S‐acylated proteins, with a proteomic coverage of almost an order of magnitude greater than any other single S‐acylproteomics study. Gene ontology analysis of the high‐confidence candidates indicates that S‐acylated proteins are highly significantly enriched in subcellular organelles including mitochondria, endoplasmic reticulum, Golgi apparatus, nucleolus, ribosome, and peroxisome, as well as in biological processes like rRNA processing, translation, and fatty acid beta‐oxidation. Interestingly, many candidate prostate cancer biomarkers, such as prostate‐specific antigen, prostate‐specific membrane antigen, and alpha‐methylacyl‐CoA racemase, are S‐acylated in LNCaP cells. This suggests that S‐acylation may play an important role in prostate cancer progression, and that the S‐acylproteome is a rich source for prostate cancer biomarker discovery. In summary, our ngPalmPISC method opens up a new avenue for proteome‐wide analysis of S‐acylated proteins in cells and clinical specimens, and its application reveals that S‐acylation is more prevalent than we previously thought.Support or Funding InformationDoD PCRP W81XWH‐15‐1‐0167Steve Spielberg Cancer Discovery Foundation (CSR205927)