Background The measurement of ileal amino acid (AA) digestibility is invasive and inappropriate when applied to vulnerable populations. The dual isotope method has been developed over the past 5 years as an alternative method. Objective The aim of this work was to compare the indispensable AA (IAA) digestibility values of two different proteins obtained using the dual isotope and the standard ileal balance methods in the same subjects. Methods Fifteen healthy adults completed the study. Over 4 h, they ingested nine successive portions of mashed potatoes containing the test protein (pea protein or casein) labeled intrinsically with 15N and 2H, and a 13C-free AA mixture as a reference for the dual isotope method. Plasma was sampled regularly over the 8-hour postprandial period, while the ileal digesta was collected continuously via a naso-ileal tube. Isotopic enrichments (15N, 13C) were measured in the digesta for the direct determination of ileal IAA digestibility, while plasma enrichments (2H, 13C) were measured to determine IAA digestibility using the dual isotope method. Results The 4-h repeated meal procedure enabled the almost complete digestion of test proteins at 8 h and the attainment of a plasma isotopic plateau between 2.5 and 4 h. These conditions were necessary to perform the ileal balance and dual isotope methods simultaneously. For pea protein, the mean IAA digestibility was similar between the two methods, but significant differences (from 10 to 20%) were observed for individual IAA values. For casein, IAA digestibility was significantly lower with the dual isotope method for all the IAA analyzed. Conclusion Under our experimental conditions, the degree of agreement between the dual isotope and ileal balance methods varied among AAs and depended on the protein source. Further research is needed to validate the dual isotope method. This study was registered at clinical trials.gov as NCT04072770.