The purpose of this study was to develop a method for simultaneous analysis of aucubin, catalpol, and geniposide, which are representative iridoid glycoside constituents of Rehmannia glutinosa, in rat plasma, urine, and feces using hydrophilic interaction ultra high-performance liquid chromatography with tandem mass spectrometry. The three components were separated using 10 mmol/L aqueous ammonium formate containing 0.01% (v/v) formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.2 mL/min, equipped with a Kinetex HILIC column (50 × 2.1 mm, 2.6 μm). Quantitation of this analysis was performed on a triple quadrupole mass spectrometer employing electrospray ionization and operated in multiple reaction monitoring mode. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma constituents. In all three iridoid glycosides, both the intra- and interbatch precisions (coefficient of variation %) were less than 4.81%. The accuracy was 96.56-103.55% for aucubin, 95.23-106.21% for catalpol, and 94.50-104.16% for geniposide. The developed analytical method satisfied the criteria of international guidance and was successfully applied to pharmacokinetic studies including oral bioavailability of aucubin, catalpol, and geniposide, and their urinary and fecal excretion ratios after oral or intravenous administration to rats. The new method was also applied to measure plasma protein binding ratios in vitro.