Background: In the present study, we identified two cytotoxic compounds from the root of Rumex crispus L. using a bioassaybased method. Methods and Results: Compared with the other fractions, the diethyl ether (Et 2 O) fraction of R. crispus root extract exhibited the strongest of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect [scavenging concentration 50% (SC 50 ) = 63.8 ± 1.47 ㎍/㎖], nitric oxide (NO) production inhibitory effect on the mouse macrophage cell line RAW264.7 [inhibitory concentration 50% (IC 50 ) = 60.9 ± 7.52 ㎍/㎖] and cytotoxicity effect on the human hepatoma cell line, HepG2 [lethal concentration 50% (LC 50 ) = 115.4 ± 1.86 ㎍/㎖]. According to the bioassay-based method, two cytotoxic compounds were purified from the Et 2 O fraction by using column chromatography and preparative high performance liquid chromatography (prep-HPLC). These two compounds were identified as parietin and chrysophanol by using nuclear magnetic resonance (NMR) and liquid chromatography quadruple time of flight mass spectrometry (LC-QTOF-MS). In addition, both parietin and chrysophanol exhibited a cytotoxicity effect on HepG2 cells, their LC 50 values were 169.1 ± 17.67 µM and 111.5 ± 6.62 µM, respectively. Conclusions: Parietin and chrysophanol isolated from the Et 2 O fraction of the R. crispus root extract showed cytotoxicity in HepG2 cell.