ObjectiveTo develop a psychometrically valid questionnaire for testing knowledge on micronutrients and to assess the relationship between knowledge and biomarkers of micronutrient status among adolescents.DesignCross-sectional, institution-based, validity and reliability study.SettingSeven higher secondary schools were covered in the limits of Greater Hyderabad Municipal Corporation, Hyderabad, India.SubjectsStudents aged 15–19 years, n 92 for the pre-test, n 108 for test–retest and n 109 for studying the relationship between knowledge and biomarkers of Fe, retinol, ascorbic acid, α-tocopherol, folic acid and vitamin B12 status.ResultsFrom an item pool of 106, thirty-one items were selected based on content validity. Statistical tools to obtain a valid and reliable questionnaire among adolescent boys and girls resulted in eighteen items with a difficulty index of 0�11–0�86, discrimination index of 0�20–0�72 and validity index (point bi-serial correlation) of 0�10–0�62. Reliability as measured by Cronbach's α was 0�71 and the intra-class correlation coefficient was 0�80. A Bland–Altman plot showed good agreement between test and retest scores. The mean response score to the eighteen-item questionnaire was 5�2 (sd 2�68). The mean values of serum retinol were significantly different (P = 0�022) between groups below (24�8 (sd 6�64) μg/dl) and above (28�0 (sd 7�67) μg/dl) the 50th percentile of knowledge score. The relationship persisted after controlling for economic status as a covariate using analysis of covariance (P = 0�018). Other micronutrients did not show any significant relationship.ConclusionsA valid and reliable eighteen-item knowledge questionnaire was constructed and found to have a significant positive relationship with plasma retinol status alone.