Many pharmacological studies have been carried out to describe multiple biological properties of Ashwagandha (Withania somnifera) and the additional safety information on repeated dose toxicity is limited. Therefore, the aim of this study was to obtain safety data for KSM-66 Ashwagandha Root Extract (ARE) through repeated-dose toxicity in Wistar rats according to the Organisation for Economic Co-operation and Development (OECD) test guideline (TG 408). ARE was orally administered to rats at doses of 0, 500, 1000, and 2000 mg/kg body weight/day for 90-day and reversibility of effects of 0 and 2000 mg/kg body weight/day was assessed for 14 days. All the animals from treated, control, recovery control and recovery groups were observed for clinical signs of toxicity once daily, detailed clinical examination every week after dosing and before necropsy day. Mortality/Morbidity was observed twice daily. In addition, observations were noted in the detailed sensory reactivity, functional assessments, body weight, food consumption, ophthalmological examination, hematological parameters, biochemical parameters, organ weights, histopathological findings. The present results show that the no observed adverse effect level (NOAEL) of KSM-66 Ashwagandha Root Extract was considered to be 2000 mg/kg body weight/day in rats after repeated oral administration for 90-day under the present study conditions.