Ashwagandha, or Indian ginseng (Withania somnifera), is of considerable importance in the Solanaceae family due to the presence of a group of steroid lactones called withanolides. Given the limited production of these metabolites in the roots and leaves of the plant under natural conditions and the high demand from the pharmaceutical industry, it is crucial to use the hairy root method to increase metabolite production. To induce bacteria under laboratory conditions, tissue culture and its various steps are typically required. However, nowadays, an alternative and simpler transgenic method is used that does not rely on tissue culture and can produce transgenic plants with an increased yield of secondary metabolites. In this experiment, Agrobacterium rhizogenes strain A4 was injected into the plant at the wound site located at the first node of the seedling. Fifteen days later, hairy roots appeared, and after the growth of the transgenic plants in the greenhouse, growth characteristics (such as root volume and root area) were measured, and phytochemical analyses were performed on both transgenic and control plants. The experiment followed a completely randomized design with 3 replications. The transgenic plants outperformed the controls in terms of agronomic traits: root length (1.12 times longer), root volume (2 times greater), and root area (1.49 times larger). As a result of this study, catalase enzyme activity and flavonoid levels were found to be lower in transgenic plants than in control plants. Correlation results showed that catalase enzyme activity and flavonoid content had positive and significant correlations with root length, volume, and area. Based on the findings of this research, the transgenic method, without the need for tissue culture, can be introduced as a useful approach in this plant to increase root hair production without increasing antioxidant enzyme activity.