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Abstract: A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the quantitation of 6-keto PGF1α in human urine and plasma. Prostacyclin (PGI2) is a locally acting prostanoid, which mediates vasorelaxation and inhibition of platelet aggregation. 6-Keto PGF1α is the most-immediate metabolite of PGI2. Samples were spiked with an internal standard (6-keto PGF1α-d4), purified by immuno-affinity chromatography and selected reaction monitoring (SRM) was performed. Analytical validation of the 6-keto PGF1α assay was performed in urine. This included an assessment of assay precision, recovery, stability, sensitivity and linearity. Urinary 6-keto PGF1α concentrations were also correlated to urinary 2,3-dinor-6-keto PGF1α (PGIM) concentrations using urine samples collected from 16 healthy volunteers. The mean concentration of 6-keto PGF1α in urine (mean±SD) was 92±51pg/ml or 168±91pg/mg creatinine. Overall, there was a statistically significant correlation between urinary 6-keto PGF1α and PGIM (r 2 =0.55, p ≤0.001; slope=2.7; y-intercept=130). However, PGIM was approximately 3-fold more abundant than 6-keto PGF1α in urine. In addition, 6-keto PGF1α concentrations were measured in EDTA plasma samples obtained from 7 healthy donors. The mean concentration of 6-keto PGF1α in plasma was 1.9±0.8pg/ml (±SD). [Copyright &y& Elsevier]