Background: Infant formula (IF) has to provide at least the same amount of amino acids (AAs) as human milk (HM). AA digestibility in HM and IF was not studied extensively, with no data available for tryptophan digestibility. Objectives: The present study aimed to measure the true ileal digestibility (TID) of total nitrogen and AAs in HM and IF to estimate AA bioavailability using Yucatan mini-piglets as an infant model. Methods: Twenty-four 19-day-old piglets (males and females) received either HM or IF for 6 days or a protein-free diet for 3 days, with cobalt-EDTA as an indigestible marker. Diets were fed hourly over 6 h before euthanasia and digesta collection. Total N, AA, and marker contents in diets and digesta were measured to determine the TID. Unidimensional statistical analyses were conducted. Results: Dietary N content was not different between HM and IF, while true protein was lower in HM (À4 g/L) due to a 7-fold higher nonprotein N content in HM. The TID of total N was lower (P < 0.001) for HM (91.3 AE 1.24%) than for IF (98.0 AE 0.810%), while the TID of amino acid nitrogen (AAN) was not different (average of 97.4 AE 0.655%, P ¼ 0.272). HM and IF had similar (P > 0.05) TID for most of the AAs including tryptophan (96.7 AE 0.950%, P ¼ 0.079), except for some AAs (lysine, phenylalanine, threonine, valine, alanine, proline, and serine), with small significant difference (P < 0.05). The first limiting AA was the aromatic AAs, and the digestible indispensable AA score (DIAAS) was higher for HM (DIAAS HM ¼ 101) than for IF (DIAAS IF ¼ 83). Conclusion: HM, compared to IF, had a lower TID for total N only, whereas the TID of AAN and most AAs, including Trp, was high and similar. A larger proportion of non-protein N is transferred to the microbiota with HM, which is of physiological relevance, although this fraction is poorly considered for IF manufacturing.