A major bioactive metabolite of linoleic acid formed by the action of 15-lipoxygenase-1 is 13(S)-hydroxy-cis-9, trans-11-octadecadienoic acid (13(S)-HODE). 13(S)-HODE is an important intracellular signal agent and is involved in cell proliferation and differentiation in various biological systems. Separation and quantification of 13(S)-HODE from biological materials has previously been achieved only by using radiolabeled linoleic acid as the substrate and two serially connected or two separate HPLC columns to achieve separation of 13(S)-HODE. In the current method, separation and quantification of 13(S)-HODE was achieved by use of a normal-phase HPLC and a solvent system containing hexane/isopropanol/acetonitrile/acetic acid (800/8/30/1, v/v) using isocratic elution with detection at 235nm. With the currently described method, good separation from unreacted interfering compounds and quantification for 13(S)-HODE were achieved within 35min with a minimum detection limit of 0.5ng per injection.