A sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method for determination of midazolam and its metabolite l-hydroxymidazolam in rat plasma was developed and validated.We take the method of acetonitrile precipitation to extracted the analytes and internal standard carbamazepine from plasma.The chromatographic separation was performed on a Zorbax SB-C18 column (150 × 2.1□mm,5 □ μm),using acetonitrile-0.1% formic acid as the mobile phase with gradient elution,delivered at a flow-rate of 0.4 ml/min.Electrospray ionization (ESI) source was applied and operated in positive ion mode,and selected ion monitoring (SIM) mode used to quantify midazolam and its metabolite l-hydroxymidazolam.Calibration curves were linear in the concentration ranges of 5-2000 ng/mL for midazolam and 10-2000 ng/mL for 1 -hydroxymidazolam,with a lower limit of quantification (LLOQ) of 5 ng/mL for midazolam and 10 ng/mL for 1 -hydroxymidazolam.Intra-and inter-day precision were less than 13% and the accuracy ranged from-10.7% to 9.5%.This developed method was successfully used for determination of midazolam and metabolite l-hydroxymidazolam in rat plasma for pharmacokinetic study.