Good-quality questionnaires are needed to assess the effectiveness of educational programs on undergraduate healthcare students’ knowledge, attitudes, understanding and behavior regarding evidence-based practice and evidence-informed practice. However, there is currently no available questionnaires designed to evaluate the outcomes of evidence-based practice and evidence-informed practice education. Based on the methods proposed by Streiner et al., this study developed and validated a questionnaire for assessing evidence-informed practice and evidence-based practice knowledge, attitudes, understanding, self-perceived application and use, and behavior among undergraduate preregistration students in the nursing and allied health disciplines. Seventy-three questionnaire items were developed and administered to varied groups of people, including students and academics. Principal component analysis was conducted to reduce dimensions and establish construct validity and internal consistency of the developed questionnaire. Following pilot-testing and expert review, the final validated questionnaire consists of 53 items (8 demographic items, 25 evidence-based practice items, and 20 evidence-informed practice items). The evidence-based practice and evidence-informed practice questionnaire is a valid and reliable tool to measure the outcomes of evidence-based practice and evidence-informed practice education.