Polymethoxylated flavonoids (PMFs), the particular flavonoid subclass in which all or almost all hydroxyls are capped by methylation, have high oral bioavailability and various activities. A sensitive high-performance liquid chromatography–diode array detection–electrospray ionization tandem mass spectrometry (HPLC–DAD–ESI-MS-MS) method was established to screen and identify the PMFs in the leaves of Murraya exotica. Eight PMF standards, including two polymethoxylated flavones, two polymethoxylated flavanones, two polymethoxylated chalcones and two PMF glycosides, were first to be analyzed in positive mode by collision-induced dissociation MS-MS. On the basis of the ESI-MS, characterizations were deduced, and in the results of the extracted ion chromatogram MS-MS experiment, 26 PMFs, including 18 flavones, five flavanones or chalcones and three PMF glycosides, were screened out from the complex extract of the leaves of M. exotica. Among them, 24 PMFs were hydroxylated polymethoxyflavonoids, whereas the rest were all permethoxylated PMFs. This was the first systematic report on the presence of PMFs in the leaves of M. exotica. The results indicated that the established analytical method could be adopted as a rapid, effective technique for the structural characterization of PMFs from the complex extracts of traditional Chinese medicines.