Abstract: A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of betamethasone in human plasma. The analyte was isocratically eluted on a Venusil XBP C₈ column (200 mm×3.9 mm ID, 5 μm) with methanol-water (containing 5 mmol·L^-1 ammonium formate) (80∶20) at a flow rate of 0.4 mL·min^-1, and detected with a triple quad LC-MS/MS using ESI with positive ionization. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 393.3→355.2 for betamethasone and m/z 361.3→343.2 for prednisolone (IS). Betamethasone was extracted from 0.5 mL human plasma with ethyl acetate. The average recovery is 88.24% and the low limit of quantitation (LLOQ) was 0.5 ng·mL^-1. The 3-day validation study demonstrated excellent precision and accuracy across the calibration range of 0.5-80.0 ng·mL^-1. The method was successfully applied to the pharmacokinetic study of compound betamethason injection in healthy Chinese volunteers.