A simple high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) method for the separation and characterization of impurities in the synthesis of triclabendazole has been developed. The analytical separation was achieved on a reversed-phase C18 column using acetonitrile and water (60:40, v/v) as mobile solvent at a flow-rate of 1.0 ml/min at 25 degrees C, and an UV detection at 230 nm. The on-line HPLC/ESI-MSn examinations were performed using ion trap analyzer with extraction ion chromatography (EIC) technique in positive or negative ion modes. The semi-preparative separation was performed with a reversed-phase column using methanol and water (75:25, v/v) as mobile solvent at a flow-rate of 4 ml/min at 25 degrees C, and an UV detection at 230 nm. Thus, two impurities were detected and identified as 5-chloro-6-(2,3,4-trichlorophenoxy)-2-methylsulfanyl-1H-benzoimidazole and 5-chloro-6-(2,3-dichlorophenoxy)-1-methyl-2-methylsulfanyl-1H-benzoimidazole. Meanwhile, some intermediates of impurity-1 in multi-step synthetic reactions, were tracked. Structural elucidation by 1D and 2D NMR and ESI-MSn was discussed.