The objectives of this study were to investigate the effect of growth regulators in callus induction, increase of biomass and to the yield more of cucurbitacin and cucurbitacin-E in leaf explants of Trichosanthes cucumerina L. var. cucumerina. The explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of auxins like 2,4-dichlorophenoxy acetic acid (2,4-D), [alpha]-naphthalene acetic acid (NAA), Indole butyric acid (IBA), Indole acetic acid (IAA), cytokinins like Kinetin (kn) and Benzyl adenine (BA). The different concentrations and combinations of BAP + IBA, 2, 4-D + BAP and 2, 4-D + kn increased the callus of fresh weight and dry weight. Among all concentrations and combinations, the best results revealed that leaf-derived callus cultured on 2,4-D (3.0mg-l) + kin (1.0 mg-l) produced the highest total cucurbitacins content with an optimum yield 4.9% w/w and cucurbitacin-E 2.75% w/w at third week.