A high-performance liquid chromatographic (HPLC) method has been developed for the determination of methylglyoxal (MG). When MG was treated with o-phenylenediamine and trichloroacetic acid, it was converted to a highly fluorescent new compound, 2-(2-benzimidazolyl)-3-methylquinoxaline (BIMQ) the chemical structure of which was elucidated by NMR and MS. BIMQ was analyzed by HPLC using a 4 mm×25 mm column packed with LiChrosorb Si-60 (10 μm). This method is highly sensitive and was satisfactorily applied to the measurement of MG in biological materials.