Withania somnifera (ashwagandha, WS) is widely used in traditional Indian Ayurvedic medicine. Studies indicate ashwagandha possesses antioxidant, anxiolytic, memory enhancing, antiparkinsonian, anti-venom, anti-inflammatory and antitumor properties. Present study evaluated the sub-acute toxicity of repeated dose administration of Ashwagandha root extract in wistar rats. Sub-acute toxicity of Ashwagandha was done as per the OECD-407 guidelines and was carried out for 28 days where satellite group was observed for 43 days. Wistar rats, 30 male and 30 females, were included in the study with 10 [5 M, 5 F] animals per group. Laboratory procedures were performed in accordance with CPCSEA guidelines. Animals were housed in standard laboratory conditions and were administered drugs orally- vehicle to control group and Ashwagandha 200, 400, 800 mg/kg body weight/day to study group. General parameters were noted, blood collection was done for haematological and biochemical parameters. All the animals were sacrificed, dissected and observed for gross necropsy and organs of high dose groups from control and Ashwagandha groups were sent for Histopathological examination. Gradual weight gain was observed in all the animals. No signs of intoxication and no changes in blood biochemistry were observed. Histopathological changes in organs were within normal limits. After repeated dose administration, Ashwagandha root powder extract did not show any major abnormality in a dose 5 times of the recommended human dose and above upto 800 mg/kg.