ABSTRACTGynostemma pentaphyllum, a traditional herbal tea, and its flavonoids possess antioxidant activities. This study was carried out to isolate and identify these flavonoids from G. pentaphyllum and determinate the contents of the main flavonoids and their antioxidant activities. Nine flavonoids were determinated rapidly from G. pentaphyllum using medium-pressure liquid chromatography combined with other simple chromatographic methods. And nine flavonoids were identified as rutin (1), 4′-O-methyl-kaempferol-3-O-rutinoside (2), ombuoside (3), kaempferol-3-β-D-O-rutinoside (4), isorhamnetin-3-O-β-D-rutinoside (5), quercetin-3-O-β-D-glucoside (6), isorhamnetin (7), kaempferol (8), and quercetin (9) by liquid chromatography mass spectrometry-ion trap time of flight and 13C nuclear magnetic resonance spectroscopy, respectively. All of them displayed various potent antioxidant effects against the DPPH radical and A549 cell injury by using H2O2-generate peroxyl radicals in vitro . A sensitive and specific ultra-performance liquid chromatography coupled with multiple-reaction monitoring mode was utilized to rapidly analyze the main six flavonoids of the ethanol extract of G. pentaphyllum within less than 8 min using a Waters Acquity UPLC BEH C18 column. The contents of them were from 57.11 to 12907.74 µg/g in G. pentaphyllum.