Abstract: OBJECTIVE To establish a specific, sensitive, efficient method for the quantitative determination of physalin L in rats plasma using LC-MS/MS system, and provide a detection means for its pharmacokinetic characteristic. METHODS The separation of physalin L was performed on a Waters XBridgetm-C₁₈ column (4.6 mm×150 mm, 3.5 μm) with a mobile phase consisted of 0.1% formic acid aqueous solution and acetonitrile (45:50). The assay was based on a negative MRM scan. RESULTS The linear range of the calibration curve (0.492-493 ng·mL^-1) was obtained with a good correlation coefficient. The lower limit of quantification was 0.492 ng·mL^-1. The average recovery was 99.23%-106.73% and the RSD of intra-and inter-day precision were <7.86%. The repeatability of the method was good. After intragastric administration of 2.0 mg physalin L to each rat, the mean peak time (t_max) was 0.69 h and the mean maximum concentration (C_max) was 77.48 ng·mL^-1. The area under the curve (AUC_0→t) was 280.78 ng·h·mL^-1, its t_1/2 was 2.89 h. CONCLUSION This study provides the physalin L characteristics of pharmacokinetics in rats, whitch is good for researchers on Physalis Calyx Seu Fructus (especially physalin compounds) in-depth study of pharmacological activity and pharmacokinetics.