The aim of this study was to produce intrinsically and uniformly doubly 15N-13C-labeled proteins. These proteins can be used as intrinsic tracers of dietary amino acids, both α-amino groups and carbon skeletons, during postprandial metabolic utilization. Two (Rhodes) laying hens were fed for 16 days with a standard poultry diet supplemented with 0, 0.2% or 0.4% of a mixture of 20 doubly 15N-13C-labeled AAs. A third hen was given a non-enriched diet, as the control. The eggs laid were collected over 24 days, from 3 days before to 4 days after supplementation. The 15N and 13C enrichments in proteins from white and yolk were measured by EA-IRMS and GC-C-IRMS for enrichment in individual amino acids. After 10 days of supplementation, the 15N enrichment reached an isotopic plateau at 1500 to 3000 ‰, depending on the supplementation level, in both white and yolk while the 13C enrichment was 220 to 650 ‰ in white and was 100 to 250 ‰ in yolk. The 15N enrichment was similar among the amino acids, except for the aromatic ones in which the enrichment was lower. The δ13C values were variable among amino acids in both white and yolk, ranging from 77 ‰ for tyrosine to 555 ‰ for proline with the 0.2 % supplementation level. In conclusion, the incorporation of 0.2 % labeled amino acids in the hen diet allowed us to achieve sufficient enrichment for metabolic studies. However, due to the non-homogeneity of the 13C labeling, adequate 13C enrichment of individual amino acids must be considered depending on the investigated metabolic pathway. Copyright © 2011 John Wiley & Sons, Ltd.