The incidence rate of hepatocellular carcinoma (HCC) remains high in numerous countries, including Thailand. There are numerous different lines of HCC treatment; however, various side effects and the resistance of cancer cells during treatment remain issues. At present, traditionally used herb plants have been widely used as alternatives to cancer therapy. Derris scandens is a Thai traditional herb which is commonly found in Thailand and widely used as a traditional medicine for numerous different diseases. The cytotoxicity of D. scandens ethanolic extract on a HCC cell line (HCC-S102) was determined using an MTT assay. Following treatment with D. scandens ethanolic extract, the induction of apoptosis was determined by Annexin V and dead cell assays, and then confirmed by the upregulation of cleaved poly(ADP-ribose) polymerase. Furthermore, a proteomic approach was used in order to study protein alteration upon treatment with D. scandens ethanolic extract coupled with liquid chromatography-tandem mass spectrometry analysis for protein identification. The results suggested that D. scandens ethanolic extract resulted in cytotoxicity against HCC-S102 cells, as the half-maximal inhibitory concentration values were 36.0±1.0, 29.6±0.6, and 22.6±1.5 µg/ml at 24, 48 and 72 h, respectively. Apoptotic cells were induced following treatment with D. scandens. The comparative proteomic profiles of D. scandens ethanolic extract-treated and untreated cells revealed various protein targets for anticancer activity including heterogeneous nuclear ribonucleoprotein (hnRNP) K, hnRNP A2/B1, stomatin-like 2 and GAPDH. In the present study, the anticancer activity of D. scandens ethanolic extract was demonstrated to affect the cell proliferation of HCC-S102 via an apoptotic pathway. The alteration in these proteins provides a better understanding of the mechanism of action of D. scandens, which may be a promising anticancer agent for the treatment of patients with HCC in the future.