Abstract: Background and Objectives: Lepidium sativum has been widely used to treat a number of ailments in traditional system of medicine such as; antidiabetic, hypocholesterolemic, antihypertensive, antidiarrheal, antispasmodic and laxative activities. The aim of the present study was to develop an efficient protocol for in vitro production the callus cultures from different explants of L. sativum. Detection the glucosinolates compounds in the different callus extracts. Also the cytotoxic potential of different callus extracts on human breast carcinoma (MCF-7 cell line), human lung carcinoma (A-549 cell line) and colon carcinoma (HCT-116 cell line). Followed by lipid peroxidation efficiency of promising extract. Materials and Methods: Different explants separated from L. sativum seedling were cultured on MS-medium supplemented with different 2,4-D and Kin combinations and then the production of calli was investigated on the media containing different growth regulators. Glucosinolates compounds from different callus cultures and seeds were detected by GC-MS analysis. Cell viability assay was assessed according to the mitochondrial-dependent reduction of yellow MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide) to purple formazan. Callus root extract was further investigated of their effect on lipid peroxidation inside breast tumor cell line through measure MDA (malondialdehyde) as the end product of lipid peroxidation. Results: The medium which contained 1.0 mg L–1 NAA+0.5 mg L–1 BAP achieved maximum callus fresh weights in all studied explants (cotyledon, hypocotyls, root and leaf). The GC-MS analysis revealed 13 compounds of glucosinolate hydrolysis products which were detected from in vitro callus tissues and seeds of L. sativum. The major of them was N-Cyano carbazole detected in the callus of hypocotyls extract. Callus root extract possessed the most potent cytotoxic activity against human breast carcinoma (MCF-7) cell line and moderate activity on Lung carcinoma (A-549). Other callus extracts showed week activities against all studied carcinoma cell lines. Callus root extract induce lipid peroxidation and increase the concentration of MDA. Conclusion: It could be concluded that, the medium which contained 1.0 mg NAA+0.5 mg L–1 BAP was more suitable for callus production from different explants. Glucosinolate hydrolysis products were detected in all studied callus cultures and seeds of L. sativum. Callus root extract induced most cytotoxic activity against human breast carcinoma (MCF-7) cell line.