Abstract: HPLC-MS/MS method was established for the determination of hydrocodone in human plasma. Diazepam was used as internal standard, methanol-0.5% formic acid water was used as the mobile phase for gradient elution, Agilent-C18 column (2.1×50 mm×3.5 μm) was used as analytical column. Electrospray ionization(ESI) source was applied， and multiple reaction monitoring(MRM) mode was operated in the positive mode with the monitor ions at m/z 300.2→199.1 for hydrocodone and m/z 285.0→193.0 for the internal standard， respectively. The linear calibration curve is obtained over the concentration range of 0.5—50 μg/L. The limit of quantitation is 0.5 μg/L(n=6). The inter and intra-day precision(RSD) is less than 15%. The average recoveries are above 75%. The method is proved to be specific, convenient, sensitive and suitable for pharmacokinetics of hydrocodone.