AIM: To investigate the pharmacokinetic properties of the main active components of Dalitong extract in SD rats after oral administration using UPLC-MS/MS. METHODS: An UPLC-MS/MS method was established to simultaneously detect tetrahydropalmatine, nobiletin and costunolide in the plasma and tissues of SD rats. The method was applied to investigate the pharmacokinetic characteristics and tissue distribution. RESULTS: After a single oral administration, the three active components were rapidly absorbed into the body, with a peak concentration (Cmax) of (13.73±7.50), (27.01±17.69) and (6.73±29.94) ng/mL for tetrahydropalmatine, nobiletin, and costunolide, respectively. The time to reach the peak concentration (Tmax) was (1.40 ± 0.93), (0.63 ± 0.28) and (2.38 ± 8.81) h, respectively. The area under the curve (AUC) was (80.43±40.03), (41.30±28.69) and (303.90 ± 136.69) ng·h·mL-1, respectively, and the elimination half-life (t 1/2) was (5.82 ± 2.22), (1.08 ± 0.01) and (4.95 ± 2.53) h, respectively. Multiple dosing significantly increased the exposure of the three components. There were significant differences in exposure between male and female rats, with higher exposure observed in female rats (P<0.05). The three active components were widely distributed in various tissues/organs, with the highest exposure observed in the gastrointestinal tract, followed by the liver and kidney. CONCLUSION: The UPLC-MS/MS method is accurate, sensitive, and reliable for the quantitative analysis of tetrahydropalmatine, nobiletin, and costunolide. These three active components in Dalitong extract can be absorbed, widely distributed in various tissues or organs, and rapidly metabolized/eliminated after oral administration. They are potential pharmacokinetic markers of Dalitong extract.