Abstract: Gliomas represent highly malignant brain tumors with high recurrence rates, susceptible to drug resistance and difficult to treat, therefore finding and screening new therapeutic candidates is extremely significant. In this study, we firstly used UHPLC-Q/TOF-MS method to identify the chemical constituents of Scutellaria barbata extract, and a total of 38 constituents were identified. A microfluidic chip model containing six independent U251 cell units was constructed, each U251 cell unit consisted of three parallel channels, the middle channel was used for culturing matrigel-encapsulated U251 cells, and the channel of medium or drug-containing medium was on both sides, the U251 cells formed a multicellular sphere in the matrigel, and were able to mimic the microenvironment of tumor hypoxia to a certain extent. Based on the constructed glioma microfluidic chip model and the perfusion flow mode for the screening of active components of Scutellaria barbata which had antitumor activity, the perfusion samples of Scutellaria barbata were analyzed by using UHPLC-Q/TOF-MS, and the relative concentrations of potential active components were calculated by the internal standard method, and it was found that the constituents of the samples containing scutellarin, apigenin-7-O-β-D-glucuronide, luteolin, scutellarein, naringenin and apigenin were significantly decreased (P < 0.05), suggesting that these six compounds have potential anti-glioma activity. Using CCK-8 assay, scratch assay and apoptosis assay to validate the medicinal efficacy of the six potential active components obtained from the screening, it was observed that all the six potential active components could reduce the proliferation of U251 cells, decrease the migration rate of the cells, and kill the cells by apoptosis. The sensitization of temozolomide performed by the six potential active compounds was further investigated, which indicated that naringenin, apigenin and luteolin in combination with temozolomide individually increased the sensitivity of U251 cells to temozolomide as well as decreased the cell viability. These results will provide technical and scientific evidence for treating glioma and further solving the anti-drug resistance phenomenon of glioma treatment by Scutellaria barbata and its active components.