Introduction Organ donation can be a life-saving opportunity for patients with severe organ failure. In India, the rate of organ donation of the deceased Indian population is exceptionally low. This necessitates enhancing awareness regarding organ donation among health care professionals and students, which can motivate the general population. Aim To assess and analyze the awareness level of healthcare professionals and students on organ donation in order to suggest potential strategies regarding improvement of awareness and willingness towards organ donation. Materials and methods A cross-sectional online and offline survey was conducted at the national level between July 2022 and September 2022. Indian healthcare professionals as well as students of various fraternities were included. The Snowball sampling technique was used for data collection. Statistical analysis was undertaken using the statistical package for the social sciences (SPSS) software, version 25. Results A total of 1,303 respondents were enrolled in this study. The majority of the participants were students (66.5%) residing in southern India (89.6%) pursuing medical profession (88.9%). The predominant age group was 18–24 years (73.9%). The awareness level regarding organ donation among healthcare professionals and students was 69%. Healthcare professionals and respondents aged 40 years and above had better awareness levels regarding organ donation which was reported as statistically significant ( p < 0.001). Conclusion Healthcare professionals and students awareness about organ donation need to be improved. Strategies including but not limited to prioritizing employment and education to the first relatives of the deceased organ donor should be implemented by the government to legally improve organ donation willingness. How to cite this article Chandrasekaran S, Chandrasekaran VP, Nandi D, Setty RS, Srinivasan O, Kaur KN. Assessment of Awareness Level Regarding Organ Donation among Healthcare Professionals and Students in India. Indian J Crit Care Med 2023;27(1):57–63.