We assessed the approach of using intracellular free amino acid enrichment as precursor enrichment for calculating the fractional synthetic rate of using the stable isotope tracer incorporation technique. We assumed that the true rate of protein synthesis was reflected by the rate of tracer incorporation over time divided by the plateau enrichment in protein. Isolated human fibroblasts and myocytes were cultured in medium supplemented with [15N]glycine, [15N]proline, and [d5]phenylalanine. Culture medium and cells were collected daily from d 1 to 5. A portion of cells harvested on d 5 was subcultured for an additional 3 passages to d 20. Protein enrichments in both cell types reached a plateau after 20 d of cell culture. In fibroblasts, the true protein synthesis rates were 0.74, 0.85, and 0.86%/h, using protein plateau enrichments of [15N]glycine, [15N]proline, and [d5]phenylalanine as precursor enrichments, respectively. When the corresponding intracellular free amino acid enrichments were used, protein synthesis rates were 0.76, 0.79, and 0.76%/h, respectively. Similarly, in myocytes, the true protein synthesis rates were 0.98 and 1.14%/h by protein plateau enrichments of [15N]glycine and [d5]phenylalanine, respectively. The synthesis rates were 0.94 and 1.01%/h by the corresponding intracellular enrichments, respectively. Extracellular amino acid enrichments resulted in underestimation of protein synthesis by a variable amount. We conclude that the intracellular free amino acid enrichment is an optimal surrogate for precursor enrichment to quantify protein synthesis.