Accurate assessment of dietary phosphorus intake is necessary to prevent hyperphosphatemia. The aim of this study was to evaluate the 24-h urine collection method for estimation of phosphate intake in healthy males. Two experiments, a 1-day and a 5-day loading test, were performed. After an overnight fast, subjects consumed test meals, 24-h urine collection was performed, and blood samples were obtained. In the 5-day loading test, a phosphorus supplement was orally administered on day 3. The association between the phosphorus content of test meals and urinary excretion, anthropometric indices, and blood biomarkers was analyzed to develop a more precise formula for estimating phosphorus intake. In the 1-day loading test, the standard deviation of predictive phosphorus intake, based on multiple linear regression analysis, was less than that for the phosphorus absorption rate. In the 5-day loading test, urinary phosphorus excretion was similar on days 2, 4 and 5, but was significantly higher on day 3 after phosphorus supplementation. Our results indicate that estimation of dietary phosphorus intake with the 24-h urine collection method, using the amount of phosphorus and urea nitrogen excretion, may increase the precision of short-term monitoring.