Abstract: Entecavir in human plasma was determined by high performance liquid coupled with-tandem mass spectrometry (HPLC-MS/MS). Diazepam was used as internal standard. Entecavir was separated on a Waters-Eterra MS-C₁₈ column(2.1×50 mm×5 μm). 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 278.2→152.1 for entecavir and m/z 285.0→193.0 for the internal standard， respectively. The linear calibration curve is obtained over the concentration range of 0.05—20 μg•L^-1. The limit of quantitation is 0.05 μg•L^-1(n=6). The inter and intra-day precision(RSD) is less than 15%. The average recoveries are above 75%.