The determination of oxidative stress biomarkers (OSBs) is useful for the assessment of health status and progress of diseases in humans. Whereas previous methods for the determination of OSBs in urine were focused on a single marker, in this study, we present a method for simultaneous determination of biomarkers of oxidative damage to lipids, proteins, and DNA. 2,4-Dinitrophenylhydrazine (DNPH) derivatization followed by solid phase extraction (SPE) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) allowed the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG), o- o'-dityrosine (diY), malondialdehyde (MDA), and four F2-isoprostane isomers: 8-iso-prostaglandinF2α (8-PGF2α), 11β-prostaglandinF2α (11-PGF2α), 15( R)-prostaglandinF2α (15-PGF2α), and 8-iso,15( R)-prostaglandinF2α (8,15-PGF2α) in urine. Derivatization with DNPH and SPE was optimized to yield greater sensitivity and selectivity for the analysis of target chemicals. The limits of detection of target analytes in urine were below 30 pg mL-1. The assay intra- and interday variability was below 16% of the relative standard deviation, and the recoveries of target chemicals spiked into synthetic urine were near 100%. The method was applied to the analysis of 21 real urine samples, and the analytes were found at a detection frequency of 85% for 8-PGF2α and 15-PGF2α, 71% for 11-PGF2α, 81% for 8,15-PGF2α, and 100% for diY, 8-OHdG, and MDA. This method offers simultaneous determination of multiple OSBs of different molecular origin in urine samples selectively with high accuracy and precision.