Abstract The purpose of this research was to develop a sensitive and reproducible UPLC–MS/MS method to analyze matrine, an anticancer compound, and to use it to investigate its biopharmaceutical and pharmacokinetic behaviors in rats. A sensitive and fast UPLC–MS/MS method was successfully applied to determine matrine in rat plasma, intestinal perfusate, bile, microsomes, and cell incubation media. The absolute oral bioavailability of matrine is 17.1 ± 5.4% at a dose of 2 mg/kg matrine. Matrine at 10 μM was shown to have good permeability (42.5 × 10 −6 cm/s) across the Caco-2 cell monolayer, and the ratio of P A–B to P B–A was approximately equal to 1 at two different concentrations (1 and 10 μM). Perfusion study showed that matrine displayed significant differences ( P < 0.05) in permeability at different intestinal regions. The rank order of permeability was ileum (highest, P w = 6.18), followed by colon ( P w = 2.07), duodenum ( P w = 0.61) and jejunum ( P w = 0.52). Rat liver microsome studies showed that CYP and UGTs were not involved in matrine metabolism. In conclusion, a sensitive and reliable method capable of measuring matrine in a variety of matrixes was developed and successfully used to determine absolute oral bioavailability of matrine in rats, transport across Caco-2 cell monolayers, absorption in rat intestine, and metabolism in rat liver microsomes.