Aspirin, the acetylsalicylic acid (ASA), is the most widely used medication to relieve pain, fever, and inflammation. Recent studies have revealed new benefits of aspirin, including reduction of heart attack, stroke, anti-cancer, and life-extension. Despite the profound effects of aspirin, the mechanism of its action remains to be elucidated. Here, we used a deuterium-labeled aspirin (D-aspirin) together with mass spectrometry-based acetylomic analysis, termed "DAcMS", to investigate the landscape of protein acetylation induced by aspirin. The DAcMS revealed the acetylomes of LPS-induced inflammatory BV2 cells and colon cancer HCT116 cells. The acetylation level was substantially induced upon aspirin treatment in both cell lines. In total, we identified 17,003 acetylation sites on 4,623 proteins in BV2 cells and 16,366 acetylated sites corresponding to 4,702 acetylated proteins in HCT116 cells. Importantly, functional analyses of these aspirin-induced acetylated proteins suggested that they were highly enriched in many key biological categories, which function importantly in inflammatory response. We further demonstrated that aspirin acetylates proteins through both acetyl-CoA-dependent and -independent pathways, and the accessible lysine residues at the protein surface are major acetylation targets of aspirin. Hence, our study provides the comprehensive atlas of aspirin-induced acetylome under disease conditions. These knowledge proffers new insight into the aspirin-directed acetylome and perhaps new drug target sites relevant to human cancer and inflammatory diseases. The MS data of this study have been deposited under the accession number IPX0001923000 at iProX.