The detection and characterization of low-level protein modifications in a complex system without a methodology for modification enrichment is a very challenging task. This study describes a high-resolution LC/MS-based background subtraction methodology for the unbiased detection and identification of acetaminophen-bound proteins formed in incubations with mouse liver microsomes. The microsomal incubations were conducted using both acetaminophen and [(13)C2,(15)N]acetaminophen at a drug concentration of 200 μM. After tryptic digestion and high-resolution LC/MS analysis, data from the two drug treatment groups were each background-subtracted against the other. Thus, peptide signals that were identical in both groups were effectively canceled out, and drug-bound peptide peaks, differing in masses between the groups because of the isotopic mass shift, were retained after background subtraction and became highlighted in the resultant base peak ion chromatograms. Follow-up MS/MS experiments with these drug-bound peptides led to the identification of three acetaminophen-bound proteins: microsomal glutathione S-transferase, oligosaccharyltransferase subunit ribophorin I, and argininosuccinate synthetase. These initial findings demonstrate the utility of the methodology and may shed new light on the mechanism of acetaminophen-induced hepatotoxicity. The approach is potentially applicable to similar tasks of identification of protein modifications in other complex biological systems.