This study was conducted to validate a food frequency questionnaire (FFQ) to assess vitamin K intake in clinical and research settings based on data from the Fifth Korea National Health and Nutrition Examination Survey (KNHANES V). We collected a subset of data on informative food items using the 24-hour recall method from adults aged 19 to 64 years from KNHANES V. The cumulative percent contribution and cumulative multiple regression coefficients for vitamin K intake from each food were computed. Twenty-five foods items were selected for the FFQ to assess vitamin K intake. The FFQ was validated against intakes derived from a 5-day food record (5DR) (n = 48). To assess the reliability of the FFQ, participants completed the self-administered FFQ (FFQ1) and a second FFQ (FFQ2) after a 6-month period (n = 54). Data were analyzed using Pearson's correlation coefficients, the cross-classification method, and Bland-Altman plots. Mean intakes were similar for vitamin K between the FFQ and dietary records, with significant correlations observed (r = 0.652), and cross-classification analyses demonstrated no major misclassification of participants into intake quartiles. Bland-Altman plots showed no serious systematic bias between the administrations of the two dietary assessment methods over the range of mean intakes. FFQ reliability was high, with a Pearson's correlation coefficient of 0.560. This pilot study shows promising validation and reliability evidence for the use of this FFQ, which is focused on vitamin K intake in adults, as an efficient screening tool in clinical and research settings.