BACKGROUND The use of Withania somnifera is increasing due to a number of its chemical constituents found useful for health. OBJECTIVE The present study was carried out to investigate the potential adverse effects (if any) of a standardized Withania somnifera extract (WSE) in rats following acute and sub chronic administration. METHODS The toxicity study was performed in Wistar rats by oral administration. An acute toxicity study was done at the dose of 2000 mg/kg. In the sub-acute study, Wistar rats (10/sex/group) were administered via gavage 0 (control), 500, 1000, 2000 mg/kg body weight/day of WSE for 28 days. Among two additional satellite groups, one group did not receive any drug while the second group received 2000 mg/kg/day for 28 days. At the end of study, the animals sacrificed and their body weight, hematology, serum chemistry, and histopathology evaluation was done. RESULTS In acute toxicity studies, oral LD50 of WSE in Wistar rats was greater than 2000 mg/kg body weight. Compared to the control group in sub-acute toxicity study, administration of extract did not show any toxicologically significant treatment related changes in clinical observations, ophthalmic examination, body weight gain, feed consumption, clinical pathology evaluation, and organ weight. Hematological and serum chemistry parameters were within the normal limits. Terminal necropsy did not reveal any treatment related gross or histopathological findings. CONCLUSIONS Based on this study, the no-observed-adverse-effect-level of WSE is 2000 mg/kg body weight, the highest level tested.