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A rapid and sensitive method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to simultaneously quantify hydroxyeicosatetraenoic (HETE), dihydroxyeicosatrienoic (DiHETrE), epoxyeicosatrienoic acid (EET), and prostaglandin metabolites of arachidonic acid in human plasma. Sample preparation consisted of solid phase extraction with Oasis HLB (30 mg) cartridges for all metabolites. Separation of HETEs, EETs, and DiHETrEs was achieved on an Acquity UPLC BEH C18, 1.7 mu m (100x2.1 mm) reversed-phase column (Waters Corp, Millford, MA) with negative electrospray ionization mass spectrometric detection. A second injection of the same extracted sample allowed for separation and assessment of prostaglandin metabolites under optimized UPLC-MS/MS conditions. Additionally, the endogenous levels of these metabolites in five different matrices were determined in order to select the optimal matrix for assay development. Human serum albumin was shown to have the least amount of endogenous metabolites, a recovery efficiency of 79-100% and a matrix effect of 71 - 100%. Linear calibration curves ranging from 0.416 to 66.67 ng/ml were validated. Inter-assay and intra-assay variance was less than 15% at most concentrations. This method was successfully applied to quantify metabolite levels in plasma samples of healthy control subjects receiving niacin administration to evaluate the association between niacin administration and eicosanoid plasma level response.