A reversed-phase high-performance liquid chromatogrphy (RP-HPLC) has been developed to analyze the metabolites of arachidonic acid based on the specificities of ultraviolet absorption of these various metabolites and is sensitive to the nanogram level. This procedure makes it possible to extract complex mixtures of eicosanoids efficiently with a single step and to analyze them simultaneously by RP-HPLC from biological samples using octadesylsilyl silica extraction column and PGB2 as an internal standard. The cyclooxygenase products {prostaglandin (PG)D2, PGE1, PGE2, PGF1α, PGF2α, 6-keto-PGF1α, and thromboxane B2 (TXB2)} and lipid peroxidation product, isoprostanes, of arachidonic acid were monitored by one isocratic HPLC system at 195 nm wavelength. The lipoxygenase products {leukotriene(LT)B4, LTC4, LTD4, and 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, 15-HETE} were measured by another isocratic HPLC system at 280 nm for LTs and 235 nm for HETEs. This method provides a simple and reliable way to extract and assess quantitatively the final arachidonic acid metabolites.