BACKGROUND: E-learning has emerged as a transformative approach in medical and dental education in India, offering flexibility and accessibility to students. However, there is a lack of a standardized tool to assess the multiple dimensions influencing e-learning utilization among undergraduate health profession students. This study aims to develop and validate the Comprehensive Knowledge, Attitude, and Practice Questionnaire (CKAPQ) to examine various factors affecting e-learning utilization among undergraduate medical and dental students. METHODS: The study employed a cross-sectional validation design using Zhou's Mixed Methods Model to develop and validate the CKAPQ. The initial questionnaire contained 59 items across eight theoretical factors. Content validity was assessed using the Lawshe method, and the CKAPQ survey was conducted from 2014 to 2017 among medical and dental students at a South Indian tertiary care university. Data were analyzed using exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) to validate the questionnaire structure and verify the theoretical framework. Model fit was assessed using indices such as Goodness-of-Fit Index (GFI), Adjusted Goodness-of-Fit Index (AGFI), Root Mean Square Error of Approximation (RMSEA), Normed Fit Index (NFI), Comparative Fit Index (CFI), Tucker-Lewis Index (TLI), and Standardized Root Mean Square Residual (SRMR). Construct validity and structural equation modelling (SEM) were assessed. RESULTS: A total of 960 students (61.6% females, 38.4% males) participated in the study. Through EFA, eight items were removed based on skewness and kurtosis values, and 35 items were excluded due to factor loadings < 0.5. The Bartlett's Test of Sphericity was significant (p < 0.001), and the Kaiser-Meyer-Olkin measure (0.733) indicated sampling adequacy. The final model comprised 16 items representing five constructs with factor loadings ranging from 0.557 to 0.834. The model demonstrated good reliability (Cronbach's α ranging from 0.65 to 0.78) and strong absolute fit indices (GFI = 0.982, AGFI = 0.971, SRMR = 0.063). Construct validity was supported by Composite Reliability (0.729-0.848), Average Variance Extracted (0.516-0.669), and Maximum Shared Variance (0.143-0.304). The SEM model revealed significant correlations between constructs, with PLE and PEI showing strong positive correlation (r = 0.614), while negative correlations were observed between PLE, PEI and other factors. CONCLUSION: The proposed theoretical model had eight factors, while the psychometric properties of the CKAPQ and calculations of fit revealed a five-factor model. The developed CKAPQ provides a new, standardized method to evaluate knowledge, attitudes, and practices related to E-learning among medical and dental students.