Abstract: With the modernization of Chinese medicine, the development of effective quality control method for Chinese herbs is one of the key scientific problems in the future. A method of high performance liquid chromatography-time-of-fight mass spectrometry (UPLC-Q-TOF/MS) couple with diagnostic ions under negative mode of ESI was developed for analyzing flavonoids in Glechoma longituba (Nakai) Kupr. An information dependent acquisition (IDA) mode was used to acquire high-precision mass spectrometric data for comprehensive profiling of constituents from Glechoma longituba (Nakai) Kupr. Both precursor ions and corresponding fragment ions (mass deviation are less than 5×10^-6) were simultaneously generated in negative mode. The precursor ions showing chemical formula could provide candidate compounds by querying the relevant database. Some database, such as Massbank, SciFinder, Chemspider, were used to quickly screen the possible compounds in Glechoma longituba (Nakai) Kupr. The characteristic groups and structure information were obtained by analyzing fragment ions, and that was very important to select out target ingredients form candidate compounds. The results show that a total of 35 flavonoids are identified, and 25 of which flavonoids are identified for the first time from Glechoma longituba (Nakai) Kupr. In addition to chrysin, apigenin, isoflavone, luteolin, kaempferol and quercetin, all the other compounds are flavonoid glycosides. The method can provide the technical support and material basis for pharmacodynamics research and quality control of Glechoma longituba (Nakai) Kupr.