Our objective is to develop a method to quantify lowconcentration metformin hydrochloride in beagle dog plasma using High Performance Liquid Chromatography(HPLC). HPLC is conducted on a Dikma Diamonsil C18 (150 mm×4.6 mm, 5μm) with acetonitrile-10.0 mmol/L sodium dihydrogen phosphate (7.5 mmol/L SDS, pH5.5)(32:68) as a mobile phase, column temperature 30℃, detection wavelength 235 nm, and flow rate 1.0 mL/min. The internal standard method is used. The results show that the identified characteristics of metformin hydrochloride and the internal standard are distinct. The regression equation is Y =0.686 8X +(-0022 2) r=0.999 5 and the linear range is 0.05~4.0 μg/L. The recovery rates are within 9978%~107.90%. The intraprecision RSD is less than 3.85%, and the interprecision is less than 563%. The developed method is sensitive, accurate, rapid and simple, and can be adopted to determine lowconcentration metformin hydrochloride in beagle dog plasma and in pharmacokinetic study.