The objective of this study investigated the effect of colchicine on growth and chemical composition of Withania Somnifera plant. Colchicine is currently used to improve crops and chemical compounds. The colchicine concentrations were 0.2, 0.3, and 0.5% for periods of 12, 24, and 36 hours. The results of both years showed that colchicine was significant in reducing plant growth parameters (plant height, leaf area, and fresh weight) compared with the control plants. Colchicine showed a significant increase in (branch number, dry weight, root length, root branches, root weight, and root diameter) compared to control plants. While it caused variation in the number of leaves, flowers, and fruits. Furthermore, colchicine at 0.5% application increased the total contents of phenols, but a slight adverse effect occurred with the total content of alkaloids. Moreover, the effect of time showed variation in results during the two years on the morphological and chemical responses of Withania Somnifera L. We recommend the use of colchicine to produce a high yield of the plant and elevate the therapeutic potency of the plant.