Natural 15N abundance (δ15N) varies between individual amino acids (AAs). We hypothesized that δ15N of nontransaminating and essential AAs (“source” AAs, such as phenylalanine) present in animal tissues could be used as a marker of dietary origin, whereas δ15N of transaminating AAs (“trophic” AAs, such as glutamic acid) could give more detailed insights into animal feed efficiency. Two diets based on dehydrated Lucerne pellets were tested in growing lambs, which promoted different feed efficiencies. No dietary effects were noted on δ15N of any AAs analyzed in lamb muscle. In addition, δ15N of phenylalanine was unexpectedly similar to that of glutamic acid, suggesting that δ15N of AAs is significantly derived from the metabolism of the rumen microbiota and, thus, are not suited for diet authentication in ruminants. In contrast, the δ15N of transaminating AAs facilitates an improved prediction of animal feed efficiency compared to the classical isotopic bulk N analysis.