Grape seed proanthocyanidin extract (GSPE), a type of functional food, possesses potent antioxidant activity. In this study, GSPE protected human embryonic kidney 293 (HEK 293) cells from H2O2-induced cell injury and oxidative stress in a dose-dependent manner. The key effective constituents that exerted the most potent antioxidative activity in GSPE were screened by using a modified ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) integrated 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical cation antioxidative activity analysis system. Two compounds, which were presumed to be Procyanidin B2 and Procyanidin C2, showed obvious antioxidant activity. H2O2 scavenging effect of Procyanidin B2 in HEK 293 cells was visualized in situ by a molecular imaging technique via a novel N-borylbenzyloxycarbonyl-3,7-dihydroxyphenoxazine (NBCD) fluorescent probe to detect levels of H2O2. In conclusion, the application of UPLC-Q/TOF MS integrated modified ABTS radical cation antioxidative activity analysis system and NBCD fluorescent probe successfully screened out and confirmed the antioxidative components from GSPE.