A high-performance liquid chromatography-diode array UV detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS) method was developed and validated for the simultaneous analysis of seven major constituents in Cortex Fraxini, including esculin, esculetin, fraxetin, fraxin, escuside, oleuropein and ligustroside. The contents of the seven constituents were determined by using HPLC-DAD, and the chemical structures of these constituents were identified by using HPLC-DAD-ESI-MS method. The separation was performed on an Agilent Zorbax SB-C18 column (250 mm x 4.6 mm i.d., 5 microm) with gradient elution of acetonitrile and 0.3% aqueous acetic acid within 40 min. Detection was performed at 254 nm. The calibration curves showed good linearity (r2>0.9992). The limits of detection (LOD) ranged from 1.07 to 3.19 ng/ml and limits of quantification (LOQ) ranged from 2.79 to 12.75 ng/ml, respectively. The intra- and inter-day precision was less than 5% and the accuracy was ranging from 96.49% to 103.55%. The recovery of the assay was in the range of 97.61-104.36%. The method was successfully applied to the quantification of the seven constituents in different samples of Cortex Fraxini. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of Cortex Fraxini.