Wushicha Granule, an over-the-counter-drug (OTC) prescription, consists of 19 traditional Chinese herbals medicines (CHMs), such as Chaihu, Hongcha, Chuanxiong, Houpo, and Gancao. The five however have not been effectively characterized by the quality-markers (Q-markers) system in current Pharmacopoeia. The study therefore established a novel database-aided ultra-high performance liquid chromatography-quadrupole- orbitrap mass spectrometry (UHPLC-Q- orbitrap MS/MS) strategy. The strategy has putatively identified 52 compounds from Wushicha Granule, mainly including flavonoids, saponins, alkaloid, lignins, and lactones. Especially, saponin glycyrrhetinic acid in the Granule was specifically identified as 18 ss- configuration (rather than 18a-configuration). Meanwhile, two pairs of isomers were fully discriminated, including vitexin vs isovitexin and daidzein vs 7,4'-dihydroxyflavone. 8 ss- Glycyrrhetinic acid, together with saponin saikosaponin A, alkaloid caffeine, lactone S-senkyunolide A, and lignin magnolol, were further studied using quantum chemical calculation, UV-vis spectra, and anti-counterfeiting validation experiment. In the validation experiment, they have successfully recognized 6 counterfeit Wushicha Granules, by means of a LC-MS equipped extraction software. Based on these results, 8 ss-glycyrrhetinic acid is recommended to replace the old Q-marker glycyrrhetinic acid; while saikosaponin A, caffeine, S-senkyunolide A, and magnolol are recommended as new Q-markers. These recommendations can not only recognize the counterfeits regarding Chaihu, Hongcha, Chuanxiong, Houpo, and Gancao, but also prevent the possible safety-incident. All these will greatly improve the efficiency and specificity of current Pharmacopoeia.