Objective:To investigate the changes in concentration of norisoboldine hydrochloride (NOR HCl) cream after transdermal administration in rats and evaluate transdermal drug release performance of the cream by microdialysis technique. MethodsA UPLC-MS/MS method for the determination of NOR·HCl was established, and its specificity, stability, precision and matrix effect were investigated. The NOR·HCl cream was applied onto the bare abdominal skin of the animals. The microdialysis technique and UPLC-MS/MS were used to determine the concentration of the drug, and the drug concentration-time curve was compared between NOR·HCl cream and NOR·HCl normal saline solution.  Results:Gradient elution was performed with sinomenine (m/z 330→239) as internal standard and acetonitrile -0.1% formic acid as mobile phase at a flow rate of 0.4 mL·min-1 and a column temperature of 45 ℃. It was shown in multiple reaction monitoring (MRM) that NOR·HCl (m/z 314→265) had good linearity over a range of 1～520 ng·mL-1, with R,2,=0.997, methodological RSD,<,10% and matrix effect of 86.89% - 100.80%, which met the standards. After administration of NOR·HCl cream and normal saline solution, the maximal drug concentration (C,max,) in microdialysis solution was (110.60±22.28) and (37.63±1.01) ng·mL-1 respectively and area under the concentration-time curve (AUC0-10) was (511.17±4.78) and (88.20±1.45) ng·h·mL-1 respectively. Compared with normal saline solution, NOR·HCl cream increased C,max, by 2.94 times and AUC0-10 by 5.80 times.  Conclusion:The in vivo microdialysis technique can be used to evaluate the transdermal drug release performance of NOR·HCl cream and cream can significantly increase transdermal release of NOR·HCl.