A simple, rapid and sensitive method was developed for the quantification of tenofovir in plasma of Beagle dogs using HPLC-MS/MS analysis.  The analytes tenofovir and internal standard (IS) adefovir were separated on a Zorbax SB-C₁₈ column (3.5 μm, 100 mm × 2.1 mm, Agilent, USA) with mobile phase of methanol/water containing 0.3% formic acid using a gradient elution mode at a flow rate of 0.2 mL·min⁻¹.  The plasma sample preparation was a simple deproteinization by the addition of 20% trichloroacetic acid followed by centrifugation.  The detection was performed in positive selected reaction monitoring (SRM) mode with an electrospray ionization (ESI) source.  The reactions monitored were m/z 288.1−176.2 for tenofovir and m/z 274.1−162.2 for adefovir (IS).  Linear detection responses were obtained for tenofovir ranging from 10 to 5 000 ng·mL⁻¹.  The intra- and inter-day precisions (RSD%) was no more than 6.3% with high recovery and good stability for the quantification, indicating the present method was specific, fast, accurate and reliable.  The method was successfully applied to the pharmacokinetic study of two tenofovir agents.  Tenofovir dipivoxil fumarate (BP0018, test agent) and tenofovir disoproxil fumarate (reference agent) were orally administrated to 8 Beagle dogs according to the 2×2 crossover design.  Comparing with the reference agent, the longer MRT and t_1/2z were obtained in the group of BP0018, while no significant difference was observed in AUC_0−t, AUC_0−∞, C_max and t_max between them, suggesting that tenofovir dipivoxil fumarate was bioequivalent to the tenofovir disoproxil fumarate in Beagle dogs.