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Authors’ Contributions 1,3,6 Design the study 2 Generated the idea to consider this study 4,7 Help in data collection 5 Processed and executed the designed work 6 Supervised in data analysis Article info. Received: December 10, 2018 Accepted: March 03, 2020 Funding Source: Nil Conflict of Interest: Nil Cite this article: Shaffique S, Anwer H, Asif HM, Akram M, Rehman A, Ahmed S, Ahmed K. In Vitro Evaluation of Antioxidant Activity of Homeopathic Mother Tincture and Total Phenolic Content. RADS J Pharm Pharm Sci. 2020; 8(1):26-30. *Address of Correspondence Author: Shifa.shafiquee@gmail.com Background: Antioxidants are those compounds which scavengers the free radicals, widely used in health protection and prescribed on large scale in biomedical health care units. In homeopathic system of medicine mother tinctures are prescribed. Mother tinctures of medicinal plants are having the potential to treat the various acute and chronic ailments. Objectives: The present study was accompanied to determine the total phenolic content and the antioxidant activity of homeopathic mother tincture of plant origin i.e. Baptisia tinctoria, Berberis aquifolium, Echinacea angustifolia, Hydrangea arborescens, Hydrastis canadensis, Kreosotum, and Thuja occidentalis in comparison with ascorbic acid. Methods: Antioxidant activity was evaluated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) inhibition assay method. A volume of 0.1 mM solution of DPPH was used while mother tinctures (1.25, 5, 2.5 μl volumes) were used for evaluation of antioxidant activity. Ascorbic acid was taken as a standard control and alcohol as negative control in antioxidant activity protocol. Total phenolic content was measured by Folin–Ciocalteu reagent assay. Total phenolic content of mother tinctures was measured in comparison with ascorbic acid. Results: All tested homeopathic mother tinctures of plant origin showed significant antioxidant potential except Hydrangea arborescens and Kreosotum. Alcohol which was used as negative control exhibited no antioxidant activity. Pulsatilla nigricans possess highest antioxidant potential 85±0.3 among other tested mother tinctures against DPPH free radical and 98.8±2 maximum no of phenolic contents. Conclusion: Homeopathic Mother Tinctures of plant origin showed antioxidant activity due to presence of phenolic content.