Lipid hydroperoxides are relatively stable intermediates during oxidation of fats and lipids. Lipid hydroperoxide formation during phosphatidylcholine thermal autoxidation was measured with the luminol-enhanced chemiluminescent (LCL) method. The method optimized for best noise/signal ratio provided a reliable and sensitive data up to 50 pmol/L of detected hydroperoxide. The observed chemiluminescence was calibrated with (9Z,11E,13S)-hydroperoxyoctadeca-9,11-dienoic acid (13-HPODE) as an external standard. To validate this method the spectrophotometric assay at 234 nm was compared with LCL results and high correlation was found. The presence of antioxidants inhibited chemiluminescence in a dose-dependent manner. Quercetin at low concentration, up to 2 µmol/L, whereas α-T above 20 µmol/L exhibited prooxidative properties. Possible mechanisms were discussed. The described LCL method is sensitive, fast and simple, is not specific to the lipids but may be used to determine the presence of the other types of peroxides.