24-hour recall is the dietary assessment method most frequently used to evaluate dietary intake; however, accuracy is an issue when using this method, especially in large-scale studies. The purpose of this study was to assess the validity of dietary intake estimation using one serving size. Estimates of energy and nutrients taken in over a 24-hr period based on actual intake amount (24HRAI) and based on estimates of one serving size (24HRSS) were compared. Data were analyzed using a paired t-test, Pearson’s correlation coefficients, and a cross-classification method. In male subjects, intake levels of energy, fat, vitamin C, vitamin B1, Zn, and total food measured using 24HRAI were significantly higher than those measured using 24HRSS. In female subjects, intake of carbohydrates, fiber, fat, vitamin A, vitamin C, vitamin B complexes, various minerals, and total food measured using 24HRAI were significantly lower than those measured using 24HRSS. Energy-adjusted Pearson’s correlation coefficients revealed that intake of all nutrients showed a significant positive relationship between the two measurement methods in both males and females. Cross-classification analysis revealed that 50.5~67.6% of women and 40.3~71% of men were classified in the same quartile of intake of each nutrient when comparing data from 24HRAI and 24HRSS. We conclude that using one serving size in 24-hr recall analysis was valid and therefore may be used in studies to assess food consumption in the general adult population. Also, this method can be used to classify energy and nutrient intake into quartile, which is useful in examining the association between diet and chronic diseases.