A sensitive and selective liquid chromatography-electrospray ionization tandem mass spectrometry(LC-ESI-MS/MS) was used for the simultaneous determination of metformin and glimepiride in beagle dog plasma with glipizide as internal standard(IS). After simplified protein precipitation with methanol, both the analytes and IS were chromatographed on a Zorbax CN column via gradient elution with methanol(containing 5 mmol/L ammonium acetate) and 5 mmol/L aqueous ammonium acetate as the mobile phase. Detection was performed by multiple reaction monitoring(MRM) scanning via ESI source operated in positive ionization mode. Specificity, linearity, accuracy, precision, recovery, matrix effect and stability were validated for metformin and glimepiride in beagle dog plasma. The calibration curves were linear in a concentration range of 10-10000 ng/mL for metformin and 4-4000 ng/mL for glimepiride with both correlation coefficients higher than 0.99. The recoveries obtained for the analytes and IS were all between 82.7% and 101.2%. The method exhibited excellent performance in terms of selectivity, robustness, short analytical time and simplicity of sample preparation. Finally, the proposed method was applied to a bioequivalence study of self-made bilayer tablet and commercial formulation containing 500 mg of metformin and 1 mg of glimepiride in beagle dogs.