Whole body synthesis of F2-isoprostanes, a family of cyclooxygenase-independent eicosanoids formed by free-radical catalysed peroxidation, should be best assessed by quantifying their urinary metabolites. Two methods for the quantitative determination of F2-isoprostane metabolites in human urine performing either thin-layer chromatography (TLC) (method A) or high-performance liquid chromatography (HPLC) (method B) prior to GC-tandem MS are described. Method A allows for simultaneous quantification of 8-iso-PGF2alpha, one prominent member of the F2-isoprostane family, and its major urinary metabolite, 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha. Mean excretion was found to be 223 and 506 pg/mg creatinine of 8-iso-PGF2alpha and 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha, respectively (n=14). A tight correlation existed between the urinary excretion of these two isoprostanes (r=0.86). Method B enables quantification of dinor-dihydro metabolites of various F2-isoprostanes including 8-iso-PGF2alpha. 2,3-Dinor-5,6-dihydro-8-iso-PGF2alpha was found to be an abundant dinor-dihydro F2-isoprostane metabolite. Validity of method A was proven by a combination of HPLC with TLC prior to GC-tandem MS analysis. A correlation was observed between the urinary concentrations of 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha measured by GC-MS and GC-tandem MS (r=0.84).