BACKGROUND: We used doxorubicin-based chemotherapy as a clinical model of oxidative assault in humans. METHODS: The study recruited newly diagnosed breast cancer patients (n = 23). Urine samples were collected immediately before (T0) and at 1 hour (T1) and 24 hours (T24) after i.v. administration of treatment. Measurements included allantoin and the isoprostanes iPF(2alpha)-III, iPF(2alpha)-VI, and 8,12-iso-iPF(2alpha)-VI along with the prostaglandin 2,3-dinor-iPF(2alpha)-III, a metabolite of iPF(2alpha)-III. All biomarkers were quantified using liquid chromatography-tandem mass spectrometry. RESULTS: In all subjects, the levels of the biomarkers increased at T1: allantoin by 22% (P = 0.06), iPF(2alpha)-III by 62% (P < 0.05), iPF(2alpha)-VI by 41% (P < 0.05), 8,12-iso-iPF(2alpha)-VI by 58% (P < 0.05), and 2,3-dinor-iPF(2alpha)-III by 52% (P < 0.05). At T24, the F2-isoprostanes returned to their baseline levels; the levels of allantoin continued to increase, although the T24-T0 difference was not statistically significant. CONCLUSIONS: These results indicate that urinary F2-isoprostanes are valid biomarkers and allantoin is a promising biomarker of oxidative status in humans. IMPACT: The levels of biomarkers change quickly in response to oxidative assault and can be used to monitor oxidative status in humans in response to treatments related either to generation of free radicals (chemotherapy and radiation therapy) or to antioxidants (inborn metabolic diseases and Down syndrome).