Abstract: Gut microbiota-mediated deglycosylationplays an important role in the metabolism of ginsenoside Rb₁. Thus, a lincomycin-induced gut microbiota dysbiosis rat model was selected to explored the pharmacokinetics and deglycosylation metabolism of ginsenoside Rb₁. An UPLC-MS/MS analytical method was developed to detect ginsenoside Rb₁ and its deglycosylated metabolite, Rd in rat plasma. The triple quadruple mass spectrometer was set in negative electrospray ionization mode by multiple reaction monitoring. The method was validated to meet the requirements of biological applications, by evaluating specificity, linearity, lower limits of quantification(LLOQ), precision, accuracy, matrix effect, recovery and stability. Gut microbiota dysbiosis rats were induced by oral administration of lincomycin(5 000 mg/kg)for 7 continuous days. The in vitro and in vivo results reveal that the reduced β-D-glucosidase activity significantly decreases the Rd formation rate in lincomycin-induced gut microbiota dysbiosis rats, leading to the pharmacokinetic alteration of ginsenoside Rb₁ and Rd in gut microbiota dysbiosis rats.