Abstract: An LC-MS/MS method was developed for the simultaneous determination prednisone acetate, prednisone and active metabolite prednisolone in dog plasma, and applied to a bioavailability and pharmacokinetics study of oral dose of prednisone acetate (2.0 mg·kg^-1) and prednisone (1.8 mg·kg^-1) given to Beagle dogs in a randomized, two-way crossover study. This experiment scheme was approved by the Experimental Animal Ethics Committee of Shanghai Institute of Medicine, Chinese Academy of Sciences. Dexamethason was used as internal standard. After extraction from the plasma by protein precipitation, the analytes and internal standard were separated on an HSS T₃ (50 mm×2.1 mm, 1.8 μm) column using a gradient elution procedure. The mobile phase consisted of methanol and 5 mmol·L^-1 ammonium acetate aqueous solution (0.1% formic acid). Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 401.2→295.2 for prednisone acetate, m/z 359.2→313.2 for prednisone, m/z 361.2→325.1 for prednisolone, m/z 393.2→373.0 for dexamethason. After prednisone acetate was administered, the C_max of prednisone was (25.1 ±3.61) ng·mL^-1 and AUC_0-t was (115 ±27.2) h·ng·mL^-1, while the C_max of prednisolone was (207 ±38.5) ng·mL^-1 and AUC_0-t was (760 ±218) h·ng·mL^-1. After prednisone was administered, the C_max of prednisone was (67.9 ±22.6) ng·mL^-1 and AUC_0-t was (160 ±19.3) h·ng·mL^-1, while the C_max of prednisolone was (582 ±81.4) ng·mL^-1 and AUC_0-t was (1 310 ±140) h·ng·mL^-1. The relative bioavailability of prednisone acetate to prednisone was only 57.1%.