A determination method for the antitripanosomal agent, isometamidium (IMD) in cattle muscle, liver, fat and milk has been developed by using HPLC. The drug was extracted from a sample with acetonitrile-0.25mol/L ammonium formate methanol solution, and purified by liquid-liquid partition with ethyl acetate and water, followed by extraction of the water layer in the presence of NaCl with acetonitrile. After evaporation of acetonitrile, the residue was taken up in 1mL of 0.25mol/L ammonium formate methanol solution. The drug was determined by HPLC with detection at 380nm using an octyl column (TSK-GEL Octyl-80Ts, 4.6mm i. d.×150mm), and acetonitrile-0.03mol/L citric acid containing 0.005mol/L sodium 1-heptanesulfonate (3:7) as an eluent. Recoveries of the drug from cattle muscle, liver, fat and milk were 76.0, 75.3, 88.0 and 89.0%, respectively, when 0.1μg/g of IMD was spiked. The detection limit was 0.01μg/g. The analysis of IMD by LC/MS was also investigated.