Phenylalanine hydroxylation is necessary for the conversion of phenylalanine to tyrosine and disposal of excess phenylalanine. Studies of in vivo regulation of phenylalanine hydroxylation suffer from the lack of a method to determine intra-hepatocyte enrichment of phenylalanine and tyrosine. Apo-B 100, a hepatic export protein, is synthesized from intra-hepatocyte amino acids. We designed an in vivo multi-isotope study, ¹⁵N-phenylalanine and [²H₂]tyrosine, to determine rates of phenylalanine hydroxylation from plasma enrichments in free amino acids and Apo-B100. For independent verification of Apo-B100 as a reflection of enrichment in the intra-hepatocyte pool, [1-¹³C]lysine was used as an indicator amino acid (IAA) to measure in vivo changes in protein synthesis in response to tyrosine supplementation. Adult men (n=6) were fed an amino acid based diet with low phenylalanine (9 mg.kg^-1.d^-1, 4.54 umol.kg^-1.h^-1) and seven graded intakes of tyrosine from 2.5 (deficient) to 12.5 (excess) mg.kg^-1.d^-1. Gas chromatography quadrupole mass spectrometry did not detect any tracer in Apo-B100 tyrosine. A new and more sensitive method to measure label enrichment in proteins using Isotope Ratio Mass Spectrometry demonstrated that phenylalanine hydroxylation measured in Apo-B100 decreased linearly in response to increasing tyrosine intake and reached a break-point at 6.8 mg.kg^-1.d^-1. IAA oxidation decreased with increased tyrosine intake and reached a break point at 6.1 mg.kg^-1.d^-1. We conclude: ApoB100 is an accurate and useful measure of changes in phenylalanine hydroxylation; the synthesis of tyrosine via phenylalanine hydroxylation is regulated to meet the needs for protein synthesis; and that plasma phenylalanine does not reflect changes in protein synthesis.