There are few studies evaluating awareness of Chagas disease among healthcare professionals attending migrants from Latin America or working in Chagas-endemic areas. The objective of this study was to design and validate instruments for assessing knowledge about Chagas disease among healthcare students and residents as well as students and professionals of social and other health science disciplines. Two validated scales have been developed: the 10-item Chagas Level of Knowledge Scale for healthcare professionals (ChaLKS-Medical) and the 8-item ChaLKS-Social&Health for potential aid workers from those fields. Both scales were considered adequate in terms of readability, internal consistency, construct validity, and discriminant validity. The mean number of correct answers on the ChaLKS-Medical among respondents from non-healthcare versus healthcare sectors was 1.80 versus 7.00 (P < 0.001). The scores on the ChaLKS-Social&Health also discriminated between the knowledge levels in these two groups (1.76 versus 6.78, P < 0.001). Knowledge among medical/pharmacy students and residents on the ChaLKS-Medical was acceptable and different (mean: 5.8 and 7.4, respectively; P < 0.001). Respondents' knowledge of Chagas disease was greater in those who had previously received information on the disease; this was true in both respondents from the healthcare sector (mean correct answers, ChaLKS-Medical: 7.2 versus 4.3, P < 0.001) and in potential aid workers (mean correct answers, ChaLKS-Social&Health: 5.1 versus 1.1, P = 0.001). The metric properties of both scales are adequate for their use in supporting aid operations in Chagas-endemic countries or in providing health and social care to migrant populations in non-endemic countries.