The present study aimed at identifying potential lead compounds for diabetes mellitus drug discovery. We developed a novel method involving centrifugal ultrafiltration separation subsequent liquid chromatography with quadrupole time of flight tandem mass spectrometry(LC-Q/TOF-MS/MS) determination to screen α-glucosidase inhibitors in complex Scutellaria baicalensis Georgi(SBG) extract. By adding a second filter to the screening process, the level of non-specific binding of Compounds 1, 3, 10 and 11 was significantly decreased, and the level of non-specific binding of Compounds 5 and 15 also was reduced. As a result, five flavonoids identified as baicalein, baicalein, wogonin, chrysin, and oroxylin A, were rapidly found to interact with α-glucosidase and possess potent anti-α-glucosidase activity in vitro. Specific binding of ligands to α-glucosidase was demonstrated though the proposed method and the ligands could be ranked in order of affinity for α-glucosidase, which were corresponded to the order of inhibitory activity in vitro. In conclusion, our results indicated that the developed method is a rapid and effective screening method for rat intestinal α-glucosidase inhibitors from complex herbal medicines such as SBG. The present study aimed at identifying potential lead compounds for diabetes mellitus drug discovery. We developed a novel method involving centrifugal ultrafiltration separation subsequent liquid chromatography with quadrupole time of flight tandem mass spectrometry (LC-Q / TOF-MS / MS) determination to screen α -glucosidase inhibitors in complex Scutellaria baicalensis Georgi (SBG) extract. By adding a second filter to the screening process, the level of non-specific binding of Compounds 1, 3, 10 and 11 was significantly decreased, and the level of non-specific binding of Compounds 5 and 15 also reduced. As a result, five flavonoids identified as baicalein, baicalein, wogonin, chrysin, and oroxylin A, were rapidly found to interact with α-glucosidase and possess potent anti-α-glucosidase activity in vitro Specific binding of ligands to α-glucosidase was solved though the proposed method and the ligands could be ranked in order of affinity for α-glucosidase, which were corres in conclusion, our results indicated that the developed method is a rapid and effective screening method for rat intestinal α-glucosidase inhibitors from complex herbal medicines such as SBG.