Abstract: A comprehensive qualitative analysis of flavonoids in the Tetrastigma hemsleyanum was developed by ultra-high performance liquid chromatography-quadrupole/exactive Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap MS) and mass defect filter technique (MDF). UPLC-Q-Exactive Orbitrap MS is one of the powerful tools for analyzing complex systems of Traditional Chinese Medicine (TCM). The method has two main advantages, one is the higher resolving power relative to triplestage quadrupole mass spectrometers, the other is significantly reducing analysis time. MDF is a new data processing technique for high-resolution mass spectrometry data, which enables a majority of the interfering ions to be automatically filtered, allowing the original data to be analyzed more effectively, accurately, and rapidly. UPLC-Q-Exactive Orbitrap MS was employed to generate high-resolution mass spectrometry data combined with mass data processing strategy, which could be used for rapid discovery and structural identification of complex compounds of TCM. After the identification of the compounds, verification of the structure is also critical. Mass Frontier is software for managing, evaluating and interpreting mass spectrometry and chromatographic data. With its advanced algorithms and rich inventory data, it is very simple to analyze the mass spectrum and verify the structure of compound. According to the literature of the chemical constituents of Tetrastigma hemsleyanum, the nucleus of flavonoids in the Tetrastigma hemsleyanum was determined as a template for MDF, and the mass range and mass loss range of MDF were obtained on the basis of the number and type of substituents. Subsequently, the UPLC-Q-Exactive Orbitrap MS acquisition-MDF-Orbitrap Traditional Chinese Medicine Library (OTCML) database search-Mass Frontier verification identification process was employed to conduct a comprehensive qualitative analysis of the flavonoids contained in the ethanol extract of Tetrastigma hemsleyanum. 22 flavonoids were identified by applying the UPLC-Q-Exactive Orbitrap MS-OTCML-Mass Frontier validation strategy, while 41 flavonoids were identified by applying UPLC-Q-Exactive Orbitrap MSMDF-OTCML-Mass Frontier validation strategy. The results showed that this combination technology could effectively eliminate more interfering ions and excavate more potential flavonoids. This research enriches the basic research of chemical substances and provides significant guidance for evaluating the quality of Tetrastigma hemsleyanum, which will be beneficial to the rational development of Tetrastigma hemsleyanum. Above all, the MDF screening method combined with UPLC-Q-Exactive Orbitrap MS qualitative analysis demonstrates its feasibility and unique advantages on identification of the chemical constituent of TCM.