Abstract: AimTo determine clonidine in rabbit plasma by LC-MS. MethodsThe LC-MS system consisted of Waters Alliance 2790 HPLC and Micromass ZQ-4000 MS. The HPLC was performed by using XTerra C18(150 mm×2.1 mm ID, 5 μm). The mobile phase, consisting of acetonitrile/ammonium hydrogen carbonate solution, was maintained to a flow-rate of 0.2 mL·min^-1 and the linear gradient elution was adopted. Mass spectrum was obtained by using electrospray ionization interface and the m/z of SIM was 230. ResultsThe average recovery was high and the method was reproducible. The calibration curve showed good linearity in the range of 1-80 μg·L^-1, the lowest limit of detection was 0.05 μg·L^-1. The Cmax, AUC0-t and tmax value of the pharmacokinetics parameter were (27±9) μg·L^-1, (5 352±1 121) μg·L^-1, (79±17) h. Conclusion The results demonstrated that the method had high sensitivity, good selectivity, accuracy and precision. It is used to determine the clonidine concentration in plasma. The transdermal patch can deliver clonidine to the surface of rabbit skin stably for periods of up to 1 week after a single application.