The antineoplastic activity of chloroform stem bark extract of rohituka, Aphanamixis polystachya (APE) used traditionally to treat spleen and liver tumors was evaluated in cultured HeLa cells by clonogenic and micronucleus assays. Treatment of HeLa cells with 0, 5, 10, 25, 50, 75 or 100µg/ml APE for different times caused a concentration-dependent reduction in the cell survival up to 6 h post-treatment (p<0.005) followed by a non-significant decline in the cells treated with APE for 24 h. Therefore, 6h treatment time was considered as an optimum time for APE exposure and further studies were carried out using this treatment time. Exposure of HeLa cells to different concentrations of APE resulted in a concentration-dependent decline in the cell viability and 100µg/ ml APE resulted in 72% cell death when compared with the non-drug treated control group. These results were further confirmed by clonogenic assay where, APE treatment caused a concentration-dependent decline in the clonogenicity of HeLa cells, and the surviving fraction of cells was reduced to 0.22 after treatment with 100µg/ml APE, the highest concentration screened. The inhibitory concentration (IC50) of APE was found to be 25µg/ml. The APE-induced DNA damage was estimated by micronucleus assay, where the treatment of HeLa cells with various concentrations of APE resulted in a concentration-dependent rise in the frequency of micronuclei in binucleate HeLa cells (MNBNCs) at 20, 30, and 40 h post-treatment. This increase in MNBNCs was significantly higher than the baseline frequency of MNBNCs. APE treatment also increased the frequency of HeLa cells bearing more than one micronucleus (MN) indicating higher degree of DNA damage. The frequency of MNBNC increased with scoring time and a maximum elevation in the MNBNC frequency was observed at 30 h posttreatment. The MNBNC induction and clonogenicity of HeLa cells was inversely related indicating that increased MNBNC formation resulted in a corresponding reduction in the cell survival. The cell proliferation indices (PI) declined with increasing concentration of APE that increased with screening time. Our study demonstrates that APE is able to kill HeLa cells effectively and this cell killing effect of APE may be due its ability to induce DNA damage.