Water deficit in first growth stages of melon (Cucumis melo L.) in formation of first true leaves after germination can be a factor limiting production. The first step for resolve the problem is genotypes evaluation and identification of drought tolerant melons. An effective method to achieve the goal is use of osmotic solution in tissue culture. Responses of Iranian melon landraces to drought was evaluated using sorbitol at 0.1, 0.2 and 0.4 M or polyethyleneglycol (PEG 6000) at 0.009, 0.012 and 0.015 M concentrations, and MS medium without treatment as the control. Coleoptile length, fresh and dry weight of shoots and roots, photosynthetic pigments, protein, proline, malondialdehyde (MDA) and antioxidant enzymes superoxide dismutase, guaiacol peroxidase and ascorbate peroxidase were measured. The PEG or sorbitol decreased coleoptile length, fresh weight and photosynthetic pigments, and led to enhancement of proline and MDA. Contents of protein and antioxidant enzymes was completely dependent on genotype and type and concentration of osmotic material. The in vitro culture for screening and identification of tolerant and sensitive drought genotypes could be rapid, useful and effective, with sorbitol mimicing drought better than PEG. After in vitro evaluation, the genotype responses to induced water deficit need to be confirmed under field conditions.