Compound-specific isotope analysis (CSIA) of amino acids (AAs) in consumer tissues is a developing technique with wide-ranging applications for identifying nitrogen (N) sources and estimating animal trophic level. Controlled experiments are essential for determining which dietary conditions influence variability in N stable isotopes (δ15N) trophic enrichment factors in bulk tissue (TEF bulk) and AAs (TEF AA). To date, however, studies have not independently evaluated the effect of protein quantity and quality (digestibility) on TEFs, complicating the application of AA-15N values for estimating trophic levels. We conducted a 98-d feeding experiment using five formulated isoenergetic feeds prepared with a high-quality protein source to evaluate the effect of protein quantity and quality on TEFs of liver and muscle tissues of juvenile Pacific yellowtail (Seriola lalandi), a carnivorous fish species. We decreased protein digestibility using well-established protocols that do not change AA profiles. Growth rates were higher in diets with higher protein content, and isotopic equilibrium was reached for both fish tissues and all treatments. Protein quantity and quality influenced isotope discrimination depending on tissue type and AA. In liver tissue, bulk TEFs showed a limited but significant relationship with protein quality, but did not differ with protein quantity nor quality in muscle. None of the pre-established source-AA (Lys, Met, Phe and Gly) TEFs varied significantly with protein quantity or quality in liver tissue. However, in muscle tissue TEFPhe increased significantly with protein content and decreased in response to reduced digestibility, indicating it may not serve as proxy for baseline isotopic values used to calculate trophic level. Among trophic AA, TEFLeu decreased significantly with increasing protein quantity in liver tissue, while both Leu and Ile TEFs decreased with lower protein digestibility in muscle tissue. Our results indicate that CSIA-AA in liver tissue provide more robust source and trophic AA-δ15N values than in muscle.