Longdan Xiegan Decoction (LXD) is a formulated preparation composed of 10 ingredient herbs, with iridoids as the main bioactive components. In this study, a rapid, simple and reliable method of simultaneous determination of four iridoid glycosides in LXD using high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (MS) was first developed and validated. The four iridoid glycosides references were isolated from LXD extract and purified using a preparative HPLC chromatography. The sample preparation for quantification comprised of a simple ultrasonic extraction and the satisfactory chromatographic separation of the four structurally similar iridoid glycosides was effected in less than three minutes on a CAPCELL PAK C(18) MGII column (3 microm, 100 mm x 2.0 mm), using an elution system of 10% methanol and their concentrations in different batches of LXD and ingredient herbs were simultaneously determined by HPLC-MS/MS using a multiple reaction monitoring (MRM) mode. The method was validated with respect to the overall intra- and inter-day variation (RSD less than 8%) and the limits of quantification for the four iridoid glycosides were 35, 20, 37 and 33 ng/mL, respectively.