Withania somnifera (ashwagandha) contains many biologically active constituents used for the prevention and treatment of various diseases. The aim of the current study was to evaluate the impact of Energy of Consciousness Healing Treatment (The Trivedi Effect®) on the characteristic properties 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 treatment), while another part was treated with the Energy of Consciousness Healing Treatment remotely by twenty renowned Biofield Energy Healers and defined as the Biofield Energy Treated sample. LC-MS data revealed that the retention time of the phytoconstituents remained same in the control and Biofield Energy Treated samples, whereas the peak area% i.e. the relative amount of the phytoconstituents was significantly altered. The peak area% at Rt of 5.1, 5.7, 6.4, 6.6, 6.9, 7.1, 7.4, 7.8, 7.9, 8.0, 8.2, 8.5, and 8.9 minutes of the treated sample were increased in the range of 0.92% to 14.67% compared to the control sample. On the contrary, the peak area% of the treated sample at Rt of 5.2, 5.3, 5.4, 5.6, 6.8, 8.1, 8.4, 8.6, 9.0, 9.1, and 9.2 minutes were decreased by 1.59% to 36.69% with respect to the control sample. A total of 14 withanolides such as sitoindoside IX, dihydrowithanolide D, withanolide A, withaferin A, ixocarpalactone A, withanolide S, withanolide sulfoxide, etc. were proposed with their structure from the molecular mass at m/z 605, 489, 473, 471, 505, and 992 at retention times of 6.9, 7.1, 7.9, 8.2, 8.5, and 9.3 minutes with the help of LC-MS, GC-MS and NMR data of both the control and Biofield Energy Treated samples. Consequently, the mass peak intensities of the treated sample were significantly changed in the range of -36.67% to 106% compared with the control sample at the same retention time. These findings suggest that Energy of Consciousness Healing Treatment could be advantageous 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 ashwagandha extract that might provide better therapeutic response against inflammatory diseases, immunological disorders, arthritis, stress, cancer, diabetes, sexual disorders, aging, and other chronic infections.