High-performance liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometry and photodiode array detection (HPLC-DAD-ESI-MSn) was developed to identify and characterize the flavonoids in a Chinese formulated preparation, Longdan Xiegan Decoction (LXD). In total, fifty-one flavonoids (27 flavones, 10 flavanones, 7 chalcones, 5 flavonols and 2 isoflavones) were characterized. Eighteen compounds among them including a newly detected flavonoid, naringin, from the ingredient herbs, were unambiguously determined by comparing the retention times (tR), UV spectral data and mass fragmentation behaviors with those of the reference compounds. Another thirty-three compounds were tentatively identified by referencing to the reported data of their UV and MS spectra. The ESI-MS/MS fragmentation behavior of flavones (OMe-substituted, O-glycosides, C-glycosides), chalcones, flavonols and their appropriate characteristic pathways were proposed. In negative ion ESI-MS all the flavonoids yielded prominent [M[BOND]H]− ions in the first order mass spectra. Fragmentation with a loss of mass of 15 Da (CH3), 18 Da (H2O), 28 Da (CO), 44 Da (CO2), 56 Da (2CO) and the residues of glucose and glucuronic acid observed in the MS/MS spectra were useful for aiding the structural identification of the flavonoids investigated.