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Background and aim: The introduction of rotavirus vaccine (RVV) in the universal immunization program of India is a big feat as it became the first nation in the World Health Organization (WHO) Southeast Asia region to do so. The involvement of huge numbers of frontline workers in introducing new vaccines in India and the underlined deficits in skills and knowledge require efficient capacity building programs. In view of this, limited research is available on the effectiveness of capacity-building interventions for healthcare workers. There is a dearth of studies from India measuring the “on-spot” impact of immunization trainings on healthcare workers. This study aims to assess the effectiveness of training in RVV introduction in enhancing the knowledge of the participants. Methods: The study was conducted among the participants attending two training workshops for the introduction of RVV: a state workshop in Pune and a regional workshop in Guwahati. The participants who attended the workshops and participated in both the pre- and post-test were included in the study. Real-time data was collected via Google forms pre- and post-training sessions. Results: In both workshops, a comparison of pre- and post-test scores of all questions taken together showed a significant increase in the knowledge level of the participants (p < 0.05). In Guwahati, the knowledge of the participants regarding doses of RVV, inadequate dosing, vaccine vial monitor (VVM), open vial policy, operationalization of RVV and monetary incentive increased significantly. In Pune, the knowledge of the participants regarding doses of RVV, bundling approach, schedule and dose, storage temperature for RVV, VVM, open vial policy, vaccine delivery and operationalization of RVV increased significantly after the training. Conclusion: A pre-planned and well-designed knowledge assessment tool can be used to understand the impact of training workshops in enhancing the knowledge and practical skills of the participants prior to the introduction of a new vaccine.