Improvement of culture media for mammalian cells is conducted via empirical adjustments, sometimes aided by statistical design methodologies. Here, we demonstrate a proof of principle for the use of constraints-based modeling to achieve enhanced performance of liver-specific functions of cultured hepatocytes during plasma exposure by adjusting amino acid supplementation and hormone levels in the medium. Flux balance analysis (FBA) is used to determine an amino acid flux profile consistent with a desired output; this is used to design an amino acid supplementation. Under conditions of no supplementation, empirical supplementation, and designed supplementation, hepatocytes were exposed to plasma and their morphology, specific cell functions (urea, albumin production) and lipid metabolism were measured. Urea production under the designed amino acid supplementation was found to be increased compared with previously reported (empirical) amino acid supplementation. Not surprisingly, the urea production attained was less than the theoretical value, indicating the existence of pathways or constraints not present in, the current model. Although not an explicit design objective, albumin production was also increased by designed amino acid supplementation, suggesting a functional linkage between these outputs. In conjunction with traditional approaches to improving culture conditions, the rational design approach described herein provides a novel means to tune the metabolic outputs of cultured hepatocytes. Biotechnol. Bioeng. 2009;103: 1176-1191. (C) 2009 Wiley Periodicals, Inc.