Aim: This study was planned to assess the awareness and use of rubber dam in routine practice among the dental practitioners of Navi Mumbai, India. Materials and methods: A Google document with a self-administered validated questionnaire consisting of 14 items was digitally sent to 300 dental practitioners via WhatsApp/e-mail and their responses were collected. Results: The surveyed responses revealed that 261 (88.2%) dental practitioners do not use rubber dam in their routine practice, whereas only 35 (11.8%) use it. However, about 166 (58.7%) dentists showed willingness to inculcate the use of rubber dam in their routine practice after appropriate training and knowledge, while 99 (35%) dentists were not sure of using it and 18 (6.4%) were not keen on using it at all. Moreover, 204 (72.1%) dental practitioners expressed interest in enhancing their knowledge regarding rubber dam. Conclusion: The results of this questionnaire-based research study revealed that the prevalence of use and awareness of rubber dam was very limited among the dental practitioners in Navi Mumbai, Maharashtra, India. Clinical significance: More emphasis should be given to the importance of rubber dam. Various continued dental education (CDE) programs and hands-on workshop can be conducted for dental practitioners.