To investigate the metabolism of scutellarin by intestinal bacteria in vitro, two strains of bacteria including Bacteroides sp. 21 and Veillonella sp. 10 were isolated from a human intestinal bacterial mixture and studied for their abilities to transform scutellarin into various metabolites. A highly sensitive and specific ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) with MetaboLynx™ software combined with the mass defect filtering (MDF) method provided high throughput capabilities for drug metabolism studies. Four metabolites were isolated using a 1.7 μm particle size Syncronis C 18 column using a gradient elution system and identified according to the mass fragmental mechanism, MS/MS fragment ions and relevant literature. The results of this study indicated that the metabolites of scutellarin were mainly in methylated and aglycone forms.