The development of reliable Food Frequency Questionnaires (FFQs) requires detailed information about the level and variation of dietary intake of the target population. However, these data are often limited. To facilitate the development of new high quality FFQs and validation of existing FFQs, we developed a comprehensive National Dietary Assessment Reference Database (NDARD) detailing information about the level and variation in dietary intake of people 20–70 years old in the general Dutch population. This paper describes the methods and characteristics of the population included in the NDARD database. A total of 1063 men and 985 women agreed to participate in this study. Dietary intake data were collected using different FFQs, web-based and telephone-based 24-h recalls, as well as blood and urine-based biomarkers. The baseline FFQ was completed by 1647 participants with a mean age of 51 ± 12 years, BMI of 26 ± 4 kg/m2, and energy intake of 2051 ± 605 kcal/day. The percentage of total energy intake from proteins, carbohydrates, and fats were 15 ± 2, 43 ± 6, and 36 ± 5 En%, respectively. A total of 1113 participants completed telephone-based recalls and 1783 participants completed web-based recalls. This database will enable researchers to validate existing national FFQs and to develop new high quality dietary assessment methods.