This study sought to develop and validate a general nutrition knowledge questionnaire (GNKQ) for Ugandan adults. The initial draft consisted of 133 items on five constructs associated with nutrition knowledge; expert recommendations (16 items), food groups (70 items), selecting food (10 items), nutrition and disease relationship (23 items), and food fortification in Uganda (14 items). The questionnaire validity was evaluated in three studies. For the content validity (study 1), a panel of five content matter nutrition experts reviewed the GNKQ draft before and after face validity. For the face validity (study 2), head teachers and health workers (n = 27) completed the questionnaire before attending one of three focus groups to review the clarity of the items. For the construct and test-rest reliability (study 3), head teachers (n = 40) from private and public primary schools and nutrition (n = 52) and engineering (n = 49) students from Makerere University took the questionnaire twice (two weeks apart). Experts agreed (content validity index, CVI > 0.9; reliability, Gwet’s AC1 > 0.85) that all constructs were relevant to evaluate nutrition knowledge. After the focus groups, 29 items were identified as unclear, requiring major (n = 5) and minor (n = 24) reviews. The final questionnaire had acceptable internal consistency (Cronbach α > 0.95), test-retest reliability (r = 0.89), and differentiated (p