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Zhi‐Zi‐Hou‐Po Decoction, consisting of Gardenia jasminoides Ellis, Magnolia officinalis Rehd. et Wils., and Citrus aurantium L, is a classical Traditional Chinese Medicine formula for the treatment of depression. In order to make good and rational use of this formula in the future, a sensitive, selective, and reliable ultra high performance liquid chromatography with tandem mass spectrometry method was developed for simultaneous determination of two iridoid glycosides (geniposide and genipin gentiobioside), two lignans (honokiol and magnolol), four flavonoid glycosides (isonaringin, naringin, hesperidin, and neohesperidin), the major bioactive constituents of Zhi‐Zi‐Hou‐Po Decoction, in rat plasma using paeoniflorin as internal standard. Plasma samples were pretreated by a simple protein precipitation with acetonitrile. Chromatographic separation was performed on a shim‐pack XR‐ODS C18 column (75 × 3.0 mm, 2.2 µm) using gradient elution with mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.5 mL/min. Mass spectrometric detection was conducted on a 3200 QTRAP mass spectrometry equipped with electrospray ionization source in negative ionization mode. Quantification was performed using multiple reactions monitoring mode. Calibration curves exhibited good linearity (r > 0.9947) over a wide concentration range for all analytes, and the lower limits of quantification were 10, 5, 1, 5, 1, 5, 1, and 5 ng/mL for geniposide, genipin gentiobioside, honokiol, magnolol, isonaringin, naringin, hesperidin, and neohesperidin, respectively. The intraday and interday precisions at three quality control levels were less than 12.3% and the accuracies ranged from −11.2 to 10.7%. Extraction recovery, matrix effect, and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of the eight analytes after oral administration of Zhi‐Zi‐Hou‐Po decoction to rats. [ABSTRACT FROM AUTHOR]