Abstract: The aim of this study was to develop a highly sensitive and specific LC-MS/MS method to explore the pharmacokinetic properties and absolute bioavailability of isoschaftoside in rats. Blood sampling was performed at different time points after intragastric administration of isoschaftoside(1. 5, 3. 0, 6. 0 mg/kg)and 0. 5 mg/kg by intravenous injection. Isoschaftoside was analyzed by a validated LC-MS/MS method in plasma; the pharmacokinetic parameters and absolute bioavailability were evaluated by software DAS 3. 0. The results showed that the linear concentration ranges of isoschaftoside was 1. 0- 500. 0 ng/mL(r=0. 997 6). The precision, accuracy, matrix effect, sensitivity, dilution reliability and stability met the requirements of biological sample analysis. For ig administration of isoschaftoside(1. 5, 3. 0, 6. 0 mg/kg), the pharmacokinetic parameter c_max was(109. 34±22. 87), (259. 84±95. 35)and(499. 26±288. 09)ng/mL; AUC_0-t was(310. 57±46. 18), (552. 67±207. 14)and(1 075. 03±371. 19)h ·ng/mL; t_1/2 was(2. 36±0. 22), (2. 91±0. 19)and(3. 04±0. 86)h; t_max was(1. 03±0. 25), (1. 18±0. 17)and(1. 5±0. 43)h; MRT_0-t was(11. 33±1. 53), (11. 27±1. 09)and(8. 29±0. 76)h, respectively. For iv administration of isoschaftoside(0. 5 mg/kg), the pharmacokinetic parameter AUC_0-t was(1 536±421. 3)h ·ng/mL; t_1/2 was(2. 57±0. 46)h; MRT_0-t was(9. 55±2. 37)h. Furthermore, the absolute bioavailability was 6. 73%, 5. 99%, 5. 80%, respectively. The LC-MS/MS analysis method established in this study was accurate and sensitive, so it can be applied to the pharmacokinetic study of isoschaftoside.