Objective: To determine the reliability and validity of a 10-item questionnaire, the Food Label Literacy for Applied Nutrition Knowledge questionnaire. Methods: Participants were elementary school children exposed to a 90-minute school-based nutrition program. Reliability was assessed via Cronbach alpha and intraclass correlation coefficient (ICC). Validity was assessed comparing the questionnaire's food choices using an objective metric of nutrition quality, the Overall Nutritional Quality Index (ONQI), via t test. Statistical significance was set at 0.05. Results: Four hundred ninety-nine children participated, 51% were female, and the average age was 8.6 (plus or minus 0.9) years. Cronbach alpha = 0.77 and ICC = 0.68 (between administrations) were observed. ONQI scores of correct responses were significantly higher when compared to the ONQI scores of incorrect responses (27.4 plus or minus 9.4 vs 16.2 plus or minus 9.4; P = 0.01). Conclusions and Implications: The Food Label Literacy for Applied Nutrition Knowledge questionnaire was found to be both reliable and a valid measure of food label literacy in children taught the Nutrition Detectives program. (Contains 1 table.)