AIM: To develop and evaluate the validity and reliability of a knowledge, attitude, and practice questionnaire related to vision screening (KAP-VST) among preschool teachers in Malaysia. METHODS: The questionnaire was developed through a literature review and discussions with experts. Content and face validation were conducted by a panel of experts (n=10) and preschool teachers (n=10), respectively. A pilot study was conducted for construct validation (n=161) and test-retest reliability (n=60) of the newly developed questionnaire. RESULTS: Based on the content and face validation, 71 items were generated, and 68 items were selected after exploratory factor analysis. The content validity index for items (I-CVI) score ranged from 0.8-1.0, and the content validity index for scale (S-CVI)/Ave was 0.99. Internal consistency was KR20=0.93 for knowledge, Cronbach’s alpha=0.758 for attitude, and Cronbach’s alpha=0.856 for practice. CONCLUSION: The KAP-VST is a valid and reliable instrument for assessing knowledge, attitude, and practice in relation to vision screening among preschool teachers in Malaysia.