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Glutathione is an important protein in many biological applications as a potential cancer treatment agent .In this study, glutathione was purified from cow milk whey bygelfiltration using Sephadex G-10 column .To know its ability as anticancer agent the study for the cytotoxic effect of the purified glutathione on, LS174T (human colorectal adenocarcinoma) cell line at different concentrations and different exposure time of treatment. The purified glutathione concentrations ranging (6.25 to 400) μg/ml for 24, 48 and 72 hours. The effect of glutathione was evaluated by employing MTT assay. The results revealed significant cytotoxic effect at levels (P<0.05) for all concentrations and for all exposure time as compared to untreated control cells, The inhibition rate IR% increased with raising of glutathione concentration and incubation period The highest inhibitory growth was shown at concentration( 400μg/ml) after 72hrs of exposure time it was 98% .