In this paper, a useful method for screening and analyzing the potential bioactive components in bioassay-guided fraction (SF-11) from Shaofu Zhuyu decoction was developed using human umbilical vein endothelial cell (HUVEC) extraction and high-performance liquid chromatography coupled with Q-TOF/MS spectrometry. In addition, the protective effects on HUVEC damage induced by adrenaline in vitro were also investigated. The results showed that SF-11 significantly inhibited the endothelin (ET) release and reversed the NO secretion of HUVEC (p < 0.05), and promoted the PGI(2) release of HUVEC (p < 0.05). Two effective components, paeoniflorin and typhaneoside, from SF-11 were screened and identified using live cell extract and HPLC coupled with Q-TOF/MS spectrometry. The compounds, paeoniflorin and typhaneoside, showed significantly inhibiting effects on the ET release and reversing of NO secretion of HUVEC (p < 0.05), with similar effects to SF-11, and promoting the PGI(2) release of HUVEC at the concentration of 0.208 and 0.013 micromol/mL, respectively (p < 0.05). These data indicated that the method of live cell extraction coupled with HPLC-MS technology is feasible, rapid and useful for screening and analyzing potential bioactive components from TCMs.