Withania somnifera (ashwagandha) root extract is very popular ancient herbal medicine. The objective of the study was to characterize and evaluate the impact of The Trivedi Effect®-Biofield Energy Healing Treatment (Energy of Consciousness) on phytoconstituents present in the ashwagandha root extract using LC-MS. Ashwagandha root extract was divided into two parts. One part was denoted as the control, while the other part was defined as The Trivedi Effect® - Biofield Energy Treated sample, which received Energy of Consciousness Healing Treatment remotely from eighteen renowned Biofield Energy Healers. The LC-MS analysis of the control and treated samples showed a very close retention time (Rt), indicated that the polarity of the phytoconstituents present in the root extract are same. The numbers of peaks observed in the total ion chromatograms were 28 and 29 in the control and treated samples, respectively. The change in the peak height% of the phytoconstituents in the treated sample was altered significantly within the range of -50.91% to 118.12% compared with the control sample. Similarly, the change in the peak area% of most of the phytoconstituents in the treated ashwagandha was significantly altered within the range of -54.95% to 66.95% compared with the control sample. An additional peak was appeared in the treated sample at Rt of 5.72 minutes, which was not found in the control sample. The LC-MS spectra indicated the presence of possible withanolides like -hydroxy-2,3-dihydro-withanolide F, withanolide A, withaferine A, withanone, withanolide D, ixocarpalactone A, withanolide S, thiowithanolide, etc. in both the samples. The peak are percentage (%) was altered in the identified withanolides, but withanolide sulfoxide was increased significantly by 12.44% in the treated sample compared with the control sample. These results indicated that The Trivedi Effect® - Biofield Energy Treatment might have an impact on the intrinsic physicochemical properties of the phytoconstituents present in the ashwagandha root extract. This could be the probable cause of alteration in the peak height, peak area, and appearance of a new peak in the treated sample. As a result, the concentrations of the phytoconstituents altered in the treated sample compared with the control sample. The treated ashwagandha root extract would be helpful for designing better pharmaceutical/nutraceutical formulations which might be providing a better therapeutic response against autoimmune diseases, nervous and sexual disorders, infectious diseases, antiaging, diabetes, cancer, ulcer, immunological disorders, stress, arthritis, etc.