Hepatocellular carcinoma (HCC) is the second most deadly and the sixth most common cancer worldwide. Although chemotherapy is the major treatment method for HCC patients, chemotherapeutic agents are known to have side effects or become ineffective through drug resistance mechanisms of tumor cells. For this reason, it is important to discover non-traditional, efficient and safe chemical agents to be used in treatment of HCC. Combrestatin A-4 (CA-4), is a natural product isolated from the South African bush willow tree Combretum caffrum, inhibits the polymerization of microtubules by binding to the colchicine binding site. Pyrazole derivatives of CA-4 are proved to exert an antimitotic activity in human cancer cells by inhibition of tubulin polymerization. In this study, we aimed to define possible anticancer properties of newly synthesized pyrazole derivatives through evaluation of their cytotoxic effect on HCC cell lines and to determine the molecular mechanism underlying this effect. Cytotoxic activities of pyrazole derivatives were analyzed by SRB assay and by real-time cell analyzer on HCC cell lines. Cell cycle analysis through flow cytometry and cell staining methods were used to determine the mechanism by which these derivatives were showing their anticancer effect. Among 42 tested pyrazole derivatives, 14 of them were found to have IC50 values below 3 μM. Four of these molecules were chosen to be further studied on each cell line. Flow cytometric analysis of cultured cells treated with these molecules demonstrated that two of these compounds caused time dependent cell cycle arrest at the G2/M phase and also caused apoptotic cell death in both cell lines. Results were confirmed through western blot analysis where active molecules cause PARP cleavage in both cell lines indicating apoptosis and decrease in Cdc2 (CDK1) and CyclinB1 levels addressing cell cycle arrest in G2/M phase (This study was partially supported by Gazi University research grant 02/2012-41).