The methanol extracts of the aerial parts, leaves and stem of Hypericum hookerianum were tested for in vitro cytotoxicity on selected normal and cancer cell lines and anti tumor activity using DLA cells. Cell viability and morphological changes were assessed. Among the three extracts tested, the stem extract of Hypericum hookerianum showed potent cytotoxicity against HEp-2 and RD cell lines. The $CTC_{50}$(concentration required to reduce viability by 50%) of this extract was found to be $2.02;{mu}g/ml$ for RD cell line, $10.25;{mu}g/ml$ for HEp-2 cell line and $100.06;{mu}g/ml$ for Vero cell line. In the clonogenic assay, no colony formation was observed up to a concentration of $100;{mu}g/ml$. In the short term cytotoxicity studies using DLA cells, 50% viability was observed in the concentration range of $50-100;{mu}g/ml$ for aerial parts, $100-200;{mu}g/ml$ for stem and more than $200;{mu}g/ml$ for leaf extracts of Hypericum hookerianum. In the long-term activity using HEp-2 cell line, no colony formation was observed over a concentration of 200 mg/ml for the stem extract. Hypericum hookerianum stem extract was fractionated into petroleum ether, chloroform, ethyl acetate and methanol soluble fractions. The petroleum ether and chloroform soluble fractions showed higher cytotoxic activity against HEp-2 cell line when compared to the other two fractions. The methanol stem extract of Hypericum hookerianum has the potential for further investigation in animal models to determine its anti-tumor activity and to identify its active principles.