Semen zizyphi spinosae (SZS) has been used to treat insomnia and anxiety for thousands of years. In this paper, a novel high-performance liquid chromatography coupled with the photodiode array detector/linear ion trap-MS(n) (HPLC-PDA/LTQ-MS(n)) method was established to separate and identify flavonoids from the extract of SZS. Separation was performed on an HYPERSIL C(18) column by gradient elution using CH(3)CN/H(2)O-CH(3)COOH as the mobile phase at a flow rate of 0.8 ml/min. UV spectral data, accurate molecular weights, and multi-stage MS/MS fragmentation information were obtained. Electrospray ionization/MS/MS fragmentation patterns were proposed. Nineteen flavonoid glycosides were identified or tentatively characterized based on their retention time, UV spectral data, accurate molecular weights, and mass fragmentation behavior. The method was useful for separation and identification of the flavonoid components from SZS and could be applied to other complex samples, especially for minor constituents.