Post-traumatic stress disorder (PTSD) is precipitated by exposure to severe traumatic events such as wars, natural disasters, catastrophes, or other traumatic events. Withania somnifera (WS) Dunal (family: Solanaceae) known traditionally as "Ashwaghanda" is used widely in ayurvedic medicine, and known to have positive role in neurodegenerative diseases. In this study, WS effect on impairment of memory due to PTSD was studied in animal models. Single-prolonged stress rat model, which consisted of restrain for 2 h, forced swimming for 20 min, rest for 15 min, and diethyl ether exposure for 1-2 min, was used to induce PTSD animals. The WS root powder extract was administered orally at a dose of 500 mg/kg/day. The radial arm water maze (RAWM) was used to assess spatial learning and memory. Enzymatic assays were used to evaluate changes in oxidative stress biomarkers in the hippocampus following treatments. The result showed that PTSD resulted in short- and long- term memory impairments. Administration of WS prevented this impairment of memory induced by PTSD. Furthermore, WS prevented PTSD induced changes in oxidative stress biomarker in the hippocampus. For quality assessment, the methanolic extract for WS was subjected to UHPLC analysis. A calibration curve for isowithanone as a marker compound was constructed. WS roots content of isowithanone was found to be 0.23% (w/w). In conclusion, WS administration prevented PTSD induced memory impairment probably through preserving changes in antioxidant mechanisms in the hippocampus.