Hydroxylated polymethoxyflavonoid glycosides (OH-PMFGs) are a rare kind of highly methoxylated flavone glycosides, which demonstrate many health-promoting bioactivities. Here we aim to identify OH-PMFGs from the leaves of Murraya paniculata using EIC (extracted ion chromatogram)-MS coupled with diagnostic product ions (DPIs) analytical method. Respective DPIs for glycosides of polymethoxyflavones, polymethoxyflavanones and polymethoxychalcones were obtained from the fragmentation pathways of eleven PMF (polymethoxylated flavonoid) standards. A sensitive HPLC-DAD-ESI-MS/MS was established for simultaneous qualitative and quantitative determination of the main OH-PMFGs in the extract. 54 OH-PMFGs including 49 flavone glycosides and 5 flavanone or chalcone glycosides were screened and identified from the leaves of M. paniculata. Meanwhile, the contents of three main OH-PMFGs in the extract were determined by HPLC-UV. It was the first systematic report of the presence of rare OH-PMFGs in the plants of the genus Murraya. The results indicated that the methodology developed could be employed as an effective technique for structural characterization of OH-PMFGs in complex extracts of TCMs (traditional Chinese medicines).