Introduction: Good knowledge and perception regarding iron supplementation are crucial to ensure adherence to iron therapy. This study aims to develop and validate a questionnaire on the knowledge and perception among pregnant women about oral iron consumption. Method: A self-administrated questionnaire was developed in Malay language through extensive literature search and a face and content validation process. The questionnaire validation involved two parts; Validation Study 1 included item response theory (IRT) and exploratory factor analysis (EFA) for the knowledge and perception items, respectively. Validation Study 2 comprised a repeat IRT analysis for the knowledge items and confirmatory factor analysis (CFA) for the perception items. Results: For the knowledge section, the initial 22 items in Validation Study 1 were reduced to 19 items after the repeat IRT analysis. The remaining 19 items had difficulty and discrimination parameters close to or within the acceptable values. For the perception section, the original 16 items were reduced to 14 in the EFA as two items had a factor loading ≤0.3. The CFA model showed poor fit of items (chi-square p-value <0.05; CFIrobust=0.73; TLIrobust=0.68; RMSEArobust=0.20; and SRMR=0.12). The Cronbach’s alpha for both sections were >0.7, and the intra-class correlation coefficient value in the knowledge and perception sections were 0.74 and 0.87, respectively. Conclusion: The results illustrate good psychometric properties for the knowledge items. However, further confirmatory validation is needed for the perception items. This questionnaire can be a valid and reliable assessment tool for assessing the knowledge of pregnant women regarding oral iron consumption.