INTRODUCTION: Due to the non-availability of formal training during residency for clinicians in transfusion medicine, there is a prevalent knowledge gap for transfusion practices, which leads to increased patient risks and elevated healthcare costs. There is no indigenous questionnaire available, which could be used for knowledge assessment. This study aims to develop and validate an Indigenous questionnaire to assess clinicians' knowledge of transfusion medicine and bedside transfusion practices. MATERIALS AND METHODS: The questionnaire was designed using a nominal group technique involving subject experts, considering the type of information required, themes, appropriate wording, layout, and presentation. The questionnaire consisted of 25 knowledge-based questions and 4 general questions. Piloting of the questionnaire was done to check for feasibility, validation, and reliability. Content validity was assessed by six experts using the Content Validity Index (CVI). Reliability was assessed using test-retest and split-half methods, with a sample of 56 participants. Cronbach's alpha and Kappa statistics were used to measure internal consistency and agreement, respectively. RESULTS: The questionnaire displayed acceptable feasibility with a mean difficulty score of 6.93 on a scale of 1-10, with test-retest responses showing near-perfect agreement (kappa value 0.8-0.99). All experts gave more than 70% agreement on the relevance of content, with a mean CVI of 85%. Test-retest reliability showed near-perfect agreement (Kappa 0.8-0.99, p = 0.008) and good internal consistency (Cronbach's α = 0.806). The split-half method yielded a Cronbach's α of 0.89 and an intraclass correlation coefficient of 0.88 (95% CI 0.57, 0.97, p = 0.001) Construct validity was confirmed through factor analysis. CONCLUSION: The developed indigenous questionnaire is a reliable and valid tool for assessing the knowledge of clinicians towards transfusion medicine and bedside transfusion practices. The detailed, methodical strategy used to prepare and validate the questionnaire ensures its applicability and relevance in various clinical settings and can be easily adopted by others intending to prepare similar questionnaires. The questionnaire is available with the author and, on demand, may be provided for knowledge assessment.