Background and objective: Vitamin B12 deficiency is a significant but often undiagnosed public health concern. Currently, its diagnosis relies on expensive laboratory tests with limited access. The present study aimed to develop and validate a 10-item screening questionnaire as a potential tool to identify inapparent vitamin B12 deficiency. Methods: This prospective, observational, real-world study was conducted among Indian physicians across multiple specialties. A self-administered, 10-question screening tool, B12-DETECT (Deficiency Evaluation Tool for Early Clinical Testing), was developed based on literature review and expert input, with scores ranging from 0 (indicating optimal vitamin B12 status) to 10 (indicating the most severe deficiency). It was validated using laboratory-based testing, with serum vitamin B12 levels (<200 pg/mL) considered as deficiency. The validity of the questionnaire was also assessed using sensitivity, specificity, and receiver operating characteristic (ROC) curve analysis. Statistical significance was set at p < 0.05. Results: Among 181 physicians evaluated, the screening tool identified 38.1% at risk for vitamin B12 deficiency (score =3), with commonly reported symptoms such as weakness, tiredness, fatigue, low mood, depression, while laboratory testing confirmed deficiency in 28.7%. It demonstrated moderate-to-high sensitivity (78.8%) and specificity (78.3%), with an area under the curve (AUC) of 0.77 (95% confidence interval [CI]: 0.703-0.850, p = 0.000). An optimal cut-off of 2.5 maximized the predictive value of the tool. Interpretations and conclusions: This simple questionnaire is a promising, cost-effective screening tool for health care professionals to detect vitamin B12 deficiency, particularly in resource-limited settings. Future studies should validate its applicability in diverse populations and explore its integration into primary health care screening programs.