We herein developed an ultra-performance liquid chromatography coupled with an electrospray ionization quadruple time-of-flight tandem mass spectrometry (UPLC/ESI-QTOF-MS) for the first time to characterize 12 iridoid glycosides from Fructus Ligustri Lucidi (FLL), a commonly used traditional Chinese medicinal herb for the treatment of kidney-deficiency and osteoporosis. In this study, a rapid and effective method was established to analyze iridoid glycosides, and 12 iridoid glycosides were detected and identified by high accurate MS data using Masslynx software. The results reveal that the iridoid glycosides were subjected to deglycosylation, major fragmentation, McLafferty rearrangement and ring cleavage, successively. Salidroside was firstly found in FLL. We have demonstrated UPLC/ESI-QTOF-MS is feasible to rapidly and reliably characterize the iridoid glycosides in FLL.