A sensitive and specific LC-MS/MS method was developed and validated for quantification of stereo isomeric F2-isoprostanes (F2-IsoPs) and prostaglandins (PGs) fromin human urine. The rapid extraction for F2-IsoPs and PGs from urine was achieved by polymeric weak anion solid phase extraction cartridge. The chromatographic separation, using HPLC-tandem mass spectrometry was carried out on a Hydro-RP column (250 x 2.0 mm i.d, Phenomenex, CA) consists of water and methanol:acetonitrile (1:1 v/v) (both containing 0.1% formic acid) with a flow rate of 0.2 mL/min. The method based on LC-MS/MS operating in MRM mode that allows base line separation of 8-iso-PGF2α, 8-iso-15(R)-PGF2α, and PGF2,15(R)-PGF2α and their simultaneous quantification over a linear dynamic range of 0.05- 10 ng/mL with accuracies 93-111%. Intra-assay precision and accuracies, evaluated at three concentrations levels (0.07, 0.5 and 5 ng/ml) were below 20%. This method is very useful for the analysis of individual F2-IsoPs and PGs and proved to be simple, sensitive, reproducible, faster than previous published method, and reliable to assess oxidative status and inflammatory conditions in both clinical and research studies.