A HPLC-MASS method has been developed for the characterization of secondary metabolites present in the methanol extract of honey bee pollen pallet. As a result, three new and five known compounds were identified or tentatively characterized by using ESIMS data. The chemical composition of methanol extract of pollen pallet is presented for the first time by using the LC-ESI-MS method. In conclusion, LC-MS/MS based chemical screening of diverse bioactive compounds in matrices of bee pollen pallet methanol extract would appear to be an effective approach for discovering new/known compounds. A high percentage of the detected compounds corresponded to glycosylated flavonoids and phenolic were identified.