Studies of lipid oxidation usually employ such model systems as purified fatty-acid methyl ester. While methyl oleate hydroperoxides (MOHPs) can only be readily separated from the matrix by HPLC, because of their heat-susceptibility and relative instability, these same techniques are unable to separate cis MOHP from trans isomers. The present study reports an enhanced, rapid separation method for cis and trans isomers of methyl oleate hydroxides, as well as HPLC determination of positional isomers per fraction of configuration isomer and isomer identification by gas chromatography-mass spectrometry.