Abstarct: Abstract The use of non-biological stresses such as drought stress in the production of plant secondary mextabolites in in vitro and the study of their mechanism in the pharmaceutical industry is greatly important. The aim of this study was to investigate the effect of drought stress on physiological and biochemical characteristics and the relationship between some genes related to drought stress resistance and vinblastine content in two cultivars of Catharanthus roseus. In this study, the effect of drought stress using polyethylene glycol 6000 at the levels of 0, 2.5, 5 and 10% was investigated at 24 and 72 hours in callus of two cultivars (Alba and Rosa) of Catharanthus roseus. The results of this study showed that drought stress at both sampling times in both cultivars significantly increased the content of total protein, malondialdehyde, catalase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase, superoxide dismutase and proline. Meanwhile, the gene exxpression of catalase and CrMPK3 at both sampling times and different levels of drought stress, confirmed the physiological results. Investigation of the exxpression of some genes involved in vinblastine biosynthesis pathway such as STR, T16H, DAT, PRX1 showed that the exxpression of these genes increased significantly by drought stress (especially in moderate stress) but the T16H had the least changes. Finally, vinblastine content was measured using HPLC to confirm the results of gene exxpression. The results of this section indicated a direct relationship between the exxpression of vinblastine pathway genes and vinblastine content. The results of the present study showed that, in order to increase the production of secondary mextabolite of vinblastine in in vitro, appropriate concentrations of polyethylene glycol can be used and the CrMPK3 gene may be involved in the activation of antioxidant enzymes as well as in the vinblastine biosynthesis pathway genes.