Patients undergoing major surgery represent a good model for the study of the hepatic metabolism of acetaminophen (APAP) after surgery and for the evaluation of how the detoxification process is influenced by aging. Thirty patients received intravenous APAP (1 g/6 h) for 4 days (D1–D4). Daily 24-h urinary metabolites—cysteine–APAP, mercapturate–APAP, APAP, and glucuronide and sulfate conjugates—as well as blood glutathione levels were compared with repeated-measures analysis of variance (significance, P < 0.05). Between D1 and D4, cysteine–APAP increased (308 ± 308 mg vs. 570 ± 512 mg, P = 0.005), and sulfate and glucuronide conjugates decreased (1,365 ± 1,084 mg vs. 694 ± 600 mg, P < 0.0001 and 2,418 ± 817 mg vs. 1,513 ± 1,076 mg, P = 0.011, respectively). Blood glutathione decreased (790 ± 125 vs. 623 ± 132 µmol/l, P < 0.0001. These changes increased with aging. APAP disposition after major surgery shifts toward the oxidative pathways of metabolism, and this is enhanced with aging. Supplementation with sulfur-containing amino acids should be investigated further as it might minimize the effect on antioxidant defenses, especially in older persons undergoing more extensive surgical procedures.