In vitro the production of natural diosgenin from callus culture became an alternative biotechnological approach to diosgenin production. This study describes the effect of plant growth regulators (PGRs) on callus induction and using different elicitors in vitro to induce the production of (Diosgenin) in callus culture from different parts of Balanites Aegyptiacae and study the effect of diosgenin extract on different cell lines. Root, stem, leaves callus calture of B. aegyptiacae, were raised on Murashing and skoog Medium (MS) supplemented with both NAA (Naphthaline Acetic acid) and 2,4D (2,4 – Dichlorophenoxy acetic acid) as plant growth regulators at different concentration. Then Elicitation process was applied by adding different amino acids and coconut milk at different concentrations to culture medium. Medium of root which supplemented with 300mg/Ltryptophane gave the maximum diosgenin content in callus extract using spectrophotometric method and HPLC analysis. Then the cytotoxic activity of the root callus extract supplemented with tryptophan was determined against HepG2 (human hepatocellular carcinoma cell line), A549 (Human Lung Cancer cell line) and the result were 51.26 μm/ml, 28.9 μm/ml respectively. The bio safety of extract was detected on the normal human cells and the result was 287.3 μm/ml.