Oxidized polyunsaturated fatty acids (PUFA) are associated to pathogenesis of diseases including cardiovascular and neurodegeneration. The novel products are not only biomarkers but also lipid mediators in gene regulation and signaling pathways. Herein, simultaneous quantitation of 28 products derived from nonenzymatic and enzymatic oxidation of PUFA i.e. 5-, 15-F2t -isoprostanes, 7-, 17-F2t -dihomo-isoprostanes, 7-, 17-F2t -dihomo-isofurans, 5-, 8-, 18-F3t -isoprostanes, 4-, 10-, 13-, 14-, 20-F4t -neuroprostanes, 5-, 8-, 9-, 11-,12-, 15-, 20-HETE, 4-, 7-, 11-, 14-, 17-HDHA, RvE1, and NPD1 using LC-(ESI)-QTOF-MS/MS was developed. These products were measurable in a single sample and the analytical time was relative short (~15 min). Furthermore, we showed that the use of internal standards is a requisite to normalize matrix effects and preparation loss for the quantitation. Validation assays indicated the method to be robust for plasma and mid-stream urine sample analysis in particular from those of age-related macular degeneration subjects, where the accuracy of quantitation displayed good repeatability.