An analytical method using high performance liquid chromatography (HPLC) was developed for the simultaneous determination of thiabendazole, 5-hydroxythiabendazole, albendazole sulfoxide, albendazole sulfone, albendazole 2-aminosulfone and mebendazole in livestock. These six benzimidazoles were extracted with acetonitrile under basic conditions, followed by a Sep-Pak C18 cartridge cleaning procedure. The HPLC separation was achieved on a Cosmosil 5C18 MS-II column (5 µm, 4.6 mm i.d. × 250 mm) using gradient elution with acetonitrile and 0.02M sodium dihydrogen phosphate (pH 3.3), and detection was performed with photodiode array and fluorescence detector. Good linearity was observed from the calibration plot at concentrations from 0.05 to 2.50 µg/mL. Recovery studies of the analytes were performed at 0.02, 0.10 and 1.00 ppm spiked levels. The average recovery of 5-hydroxythiabendazole and mebendazole at low concentrations ranged from 71.4 to 87.1% and ranged from 81.6 to 104.3% at other concentrations,with coefficient of variation less than 5.9%. The detection limit for benzimidazoles was between 0.005~0.030 ppm under photodiode array detection and 0.004~0.020 ppm under fluorescence detection. The coefficient of variation of intra-day and inter-day assays was lower than 1.92% and 7.22%, respectively. These results indicated that the developed method had an acceptable precision. This method was applied to detect benzimidazoles in samples of pork, swine liver, beef, lamb, bovine milk and goat milk purchased from various markets in Taipei. The results showed that none of the six benzimidazoles were detected in the thirty samples.