The methanol extracts of the aerial parts, leaves and stem of Hypericum hookerianum were testedfor in vitro cytotoxicity on selected normal and cancer cell lines and anti tumor activity using DLAcells. 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 celllines. The CTC50 (concentration required to reduce viability by 50%) of this extract was found to be2.02 mg/ml for RD cell line, 10.25 mg/ml for HEp-2 cell line and 100.06 mg/ml for Vero cell line. Inthe clonogenic assay, no colony formation was observed up to a concentration of 100 mg/ml. Inthe short term cytotoxicity studies using DLA cells, 50% viability was observed in theconcentration range of 50-100 mg/ml for aerial parts, 100-200 mg/ml for stem and more than 200mg/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, ethylacetate and methanol soluble fractions. The petroleum ether and chloroform soluble fractionsshowed higher cytotoxic activity against HEp-2 cell line when compared to the other twofractions. The methanol stem extract of Hypericum hookerianum has the potential for furtherinvestigation in animal models to determine its anti-tumor activity and to identify its activeprinciples.