Background: Leukemia is a cancer of blood cells which treated by different anticancer agents, Plants nowadays considered a new source for the development of new anticancer agent. Euphorbia Lathyris use in folklore to treat skin cancer. Objective Euphorbia lathyris seeds phytochemically investigated then examined its cytotoxic activity against leukemia cells. Materials and Methods: Euphorbia lathyris seed was extracted by maceration with different organic solvents (petroleum ether, chloroform and methanol), each organic extract submitted to phytochemical investigation and evaluated its cytotoxic activity on Leukemic blood with three different doses (50,100 and 150 µg mL¯1) and three different intervals. The data were evaluated by two-way analysis of variance (ANOVA). Results: In the present study, the major active constituents for petroleum ether and chloroform fractions were steroids and cholesterol while methanol fraction contains flavonoids as a major active constituent. Percent of inhibition for Petroleum ether and chloroform fractions showed a decrease in the percent of inhibition of leukemic cell proliferation with an increase of time but the same fractions showed an increase in the percent of inhibition with an increase of dose. Methanol fraction showed an increase in the percent of inhibition with increasing the dose and time. Conclusion: It had been concluded that Stepwise fractionation of Euphorbia lathyris offering a separation of active constituents according to polarities, this give a different cytotoxic effect to leukemia cells according to the active constituent that contain