Withania somnifera (Ashwagandha) root extract is very popular ancient herbal medicine. The current study was designed to investigate the impact of The Trivedi Effect® – Energy of Consciousness Healing Treatment on the structural characterization of the ashwagandha root extract using LC-MS, GC-MS, and NMR spectroscopy. Ashwagandha root extract was divided into two parts – one part was control, without any Biofield Energy Healing Treatment, while another part was treated with the Biofield Energy Healing Treatment remotely by eighteen renowned Biofield Energy Healers and defined as the Biofield Energy Treated sample. The retention time of the phytoconstituents remained same in both the control and treated samples, whereas the peak area% at respective retention time was significantly altered. The peak area% of the treated sample at Rt of 5.35, 5.55, 5.94, 6.25, 6.63, 6.76, 7.92, 8.04, 8.60, 8.73, and 9.31 min were significantly reduced by 6.15% to 60.67% compared with the control sample at Rt of 5.43, 5.65, 5.95, 6.29, 6.76, 6.85, 8.03, 8.14, 8.68, 8.78, and 9.30 min. Consequently, the peak area% of the treated sample at Rt of 7.25, 7.30, 8.27, and 8.47 min were significantly increased by 26.32%, 7.99%, 16.93% and 7.97% compared with the control sample at Rt of 7.37, 7.41, 8.36, and 8.55 min, respectively. A total of 13 withanolides were proposed with their structure from the deduced molecular mass at m/z 470, 472, 488, 504, 782, and 991 through LC-MS, GC-MS, 1H and 13C NMR analysis of both the control and treated samples. Viscosa lactone B, 27-hydroxy withanolide A, (20S, 22R)-α, 6α-epoxy-β, 5β, 27-trihydroxy-1-oxowitha-24-enolide, (20S, 22R)-4β, 5β, 6α, 27-tetrahydroxy-1-oxo-with-2, 24-dienolide were proposed in the control and treated samples at Rt of 6.85 and 7.30 min, respectively. Dihydrowithanolide D was only identified in the control sample at Rt of 7.41 min, whereas withanoside IV or withanoside VI was only present in the Biofield Treated sample at Rt of 6.76 min. Withanolide A, withaferin A, withanone, withanolide D, ixocarpalactone A and withanolide sulfoxide were found in both the control and treated samples. These findings suggest that The Trivedi Effect® – Energy of Consciousness Healing Treatment could be beneficial for altering the concentration of the phytoconstituents in the ashwagandha root extract by modifying their intrinsic physicochemical properties, which might be helpful to improve the bioavailability of active constituents of W. somnifera extract that might provide better therapeutic response against inflammatory diseases, immunological disorders, stress, arthritis, cancer, diabetes, sexual disorders, aging and other chronic infections.