In this study, a sensitive and robust ultra-performance liquid chromatography-mass spectrometry method with multiple-reaction monitoring mode was developed, validated, and applied to determine pharmacokinetics of catalpol and acteoside in normal and doxorubicin-induced chronic kidney disease rats after oral administration of Rehmannia glutinosa extract. The lower limits of quantification for catalpol and acteoside in rat plasma were 2.62 and 0.61 ng/mL, with a signal-to-noise ratio of ≥10. Precision and accuracy studies showed that catalpol and acteoside plasma concentrations were within the 10% range in all studies. The extraction recoveries of catalpol and acteoside were both >68.24% and the matrix effects ranged from 96.59 to 101.62%. The method was successfully applied to the pharmacokinetic study of catalpol and acteoside after oral administration of RG extract to normal and model rats, respectively. This study might further support the traditional use of RG to treat kidney diseases clinically.