Orthosiphon stamineus Benth. (OS) is a traditional folk medicine for the treatment of kidney stones and other urinary tract diseases. In this study, a rapid and sensitive Ultra high-performance liquid chromatography (UHPLC)-MS/MS approach was established and validated for the simultaneous quantification of nine bioactive components in rat plasma. The nine components from OS extract detected in rat plasma were danshensu, protocatechuic acid, caffeic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, cichoric acid, sinensetin and eupatorin. After liquid-liquid extraction with ethyl acetate, the plasma samples were subjected to a triple quadrupole mass spectrometer employing electrospray ionization (ESI) technique and operating in multiple reaction monitoring (MRM) with both positive and negative ion modes. The standard curves showed good linear regression (r > 0.9915) over the concentration range for the nine analytes. The inter-day and intra-day precision and accuracy were found to be within 15% of the nominal concentration. The recovery and stability of nine compounds were all demonstrated to be within acceptable limits. The approach was successfully applied to investigate the pharmacokinetic analysis of the nine bioactive components after oral administration of OS extract in rats.