Xenobiotic metabolism in the liver can give rise to reactive metabolites that covalently bind to proteins, and determining which proteins are targeted is important in drug discovery and molecular toxicology. However, there are difficulties in the analysis of these modified proteins in complex biological matrices due to their low abundance. In this study, an analytical approach was developed to systematically identify target proteins of acetaminophen (APAP) in rat liver microsomes (RLM) using two-dimensional chromatography and high-resolution tandem mass spectrometry. In vitro microsomal incubations, with and without APAP, were digested and subjected to strong cation exchange (SCX) fractionation prior to reverse-phase UHPLC-MS/MS. Four data processing strategies were combined into an efficient label-free workflow meant to eliminate potential false positives, using peptide spectral matching, statistical differential analysis, product ion screening, and a custom-built delta-mass filtering tool to pinpoint potential modified peptides. This study revealed four proteins, involved in important cellular processes, to be covalently modified by APAP. Data are available via ProteomeXchange with identifier PXD002590.