Objective：To set up a liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method for determination of betamethasone in rabbit plasma.Methods: Betamethasone and the internal standard A0 were extracted from rabbit plasma by liquid-liquid extraction with diethyl ether-hexane (41,V/V). Chromatographic separation was performed on a Zorbax Eclipse XDB-C18 column with a mobile phase consisted of acetonitrile-5 mmol/L ammonium acetate-formic acid (80200.1,V/V/V) at a flow-rate of 0.60 ml/min. A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive ion mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 393m/z 373 and m/z 393m/z 355 for betamethasone,and m/z 489m/z 357 for the internal standard. Results: The linear calibration curves were obtained within the concentration range of 15.0- 1 000 pg/ml. The lower limit of quantification was 15.0 pg/ml. The intra- and inter-day relative standard deviation over the entire concentration range was less than 13.0%. The accuracy was in the range of -1.4% to -0.6% in terms of relative error. The method was applied to a pharmacokinetic study of betamethasone dipropionate cream in rabbits. Maximal betamethasone plasma level was observed after (11.0±5.3) h and the Cmax was (167±28) pg/ml,AUC0-t was (4.24±1.68) ng·h·ml-1 after percutaneous administration of 0.5 g betamethasone dipropionate. Conclusion: This method is selective and sensitive,and can be used for the purpose of the pharmacokinetic study of betamethasone dipropionate cream.