Objective: To evaluate the effectiveness of Azadirachta indica based Herbal mouthwash to treat tooth sensitivity in patients. Method: This single-blinded clinical trial was performed at School of dentistry, Shaheed Zulfiqar Ali Bhutto Medial University, Islamabad from 1st February, 2023 to 30th April, 2023. In this interventional study incorporated 120 participants with clinically visible signs of erosion, abrasion or recession. Visual Analog Scoring (VAS) Tool was used to investigate tooth sensitivity in these patients. Values of VAS for tooth sensitivity was calculated by exposing teeth of these patients to cold air blasting with triple syringe attached to dental unit at psi-30.0 pressure between 23±30ºC for duration of one second without using Azadirachta indica based Herbal mouthwash. Later on, these patients were provided with this Herbal mouthwash and its usage was recommended twice a day for one month. After One month, tooth sensitivity of patients was determined by VAS again. Data was analyzed by Paired T-test at 95% confidence and significance < 0.05. Results: VAS mean value for tooth sensitivity of patients before using Azadirachta indica based Herbal mouthwash was higher and found to be 55.43% ± 12.04 whereas its mean value after using Herbal mouthwash for one month reduced to 35.38% ± 11.62 which was statistically significant (p value=0.001). Reduction in tooth sensitivity of patients was almost 20.05% just after one month. Conclusion: Azadirachta indica based Herbal mouthwash was potent enough to reduce the tooth sensitivity in patients after one month of its usage. doi: https://doi.org/10.12669/pjms.40.10.9826 How to cite this: Mansoor A, Mansoor E, Amir N, Hussain K. Clinical efficacy of Azadirachta indica based herbal mouthwash in treating the hypersensitivity of teeth. Pak J Med Sci. 2024;40(10):2331-2335. doi: https://doi.org/10.12669/pjms.40.10.9826 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.