Radix Isatidis (syn. Isatis indigotica Fort.) is employed in the treatment of fever, influenza, acute tonsillitis, viral hepatitis, and COVID-19, demonstrating diverse pharmacological activities, including antibacterial, antiviral, antioxidant, anticancer, and immune-regulatory effects. Furthermore, there is significant potential for the development of new clinical applications. In order to investigate the pharmacokinetic characteristics, a quantitative method for determining R,S-goitrin in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. The established LC-MS/MS method was employed to investigate the pharmacokinetics, tissue distribution, plasma protein binding, excretion, and metabolic characteristics of Radix Isatidis extract following oral administration in rats. Meanwhile, by comparing with the oral monomer group, the absorption profile of the extract in rats was assessed. After oral administration of different doses of Radix Isatidis extract (0.1,0.3,1 g/kg) to male rats, showed dose-dependent increases in R,S-goitrin's Cmax and AUC, with bioavailability at 56.33 %. No gender differences in pharmacokinetics (PK) were observed. Compared with the monomer R,S-goitrin (0.03 mg/kg), it was observed higher in vivo exposure AUC(0-t) and peak concentration Cmax of R,S-goitrin after dosing of the Radix Isatidis extract with equal dosage of R,S-goitrin. R,S-goitrin was widely distributed in immune organs (adrenal glands, thymus, lymph nodes), liver, spleen, and gastrointestinal tract after oral administration of Radix Isatidis extract (0.1 g/kg) in rats. R,S-goitrin was primarily excreted via urine, accounting for 56 % of the administered dose, with plasma protein binding ranging from 13 % to 16.4 % across different species. These findings provide data to support Radix Isatidis clinical use in antibacterial, anti-inflammatory, anticancer therapies, and formulation development.