Sleep is normally a period of relaxation and repair, important for the maintenance of physiological homeostasis and psychological balance. "Globally, millions of people experiences sleep deprivation daily". Sleep deprivation (SD) impairs cognitive functions, decreases anti-oxidative defense and induces neuronal changes. Withania somnifera (WS), commonly known as an "Indian Ginseng" has broad therapeutic applications, including anti-inflammatory activities, actions on immune system, circulatory system, central nervous system etc., The study is aimed to assess effect of Withania somnifera on antioxidant status and neurotransmitter level in sleep deprivation induced male Wistar albino rats. The study was done in the Department of Physiology, Meenakshi Medical College and Hospital, Enathur, Kanchipuram. 24 male adult Wistar rats weighing 120-150g were used for the study. They were divided into 4 groups with 6 animals in each group. (Group I - cage control, Group II - large platform control, Group III - sleep deprived group and Group IV - WS treated SD group). Animals were deprived sleep for one week using a modified multiple platform method. Oxidative stress parameters and antioxidant enzymes were measured using spectrophotometry. Neurotransmitters such as dopamine and serotonin concentration in the serum were measured by ELISA method. There was a marked (by one-way ANOVA test) decrease observed in the antioxidants enzymes in the cortex of both large platform control and sleep deprivation induced group. The group treated with W. somnifera root extract significantly reduced the free radical production and lipid peroxidation with simultaneous increase in the level of antioxidant enzymes compared to the untreated group. Also in our study the concentration of dopamine and serotonin was found to be significantly reduced (p < 0.05) in sleep deprived (SD) and large platform control group when compared to cage control group. Whereas the group treated with W. somnifera (400mg/kg b.wt) increased the neurotransmitter levels significantly. Withania somnifera proved to be an effective therapeutic agent by maintaining the antioxidant status and neurotransmitter levels.