The effect of age on the in vitro binding of valproic acid (VPA) to serum proteins was investigated in rats ranging in age from 14 days (preweanling) to 24 months (senescent). The influence of free fatty acid (FFA) and total protein (TP) concentrations on age-related changes in binding was examined. The protein binding of VPA was altered during development and aging. The VPA fraction unbound (fu) at low VPA concentrations was significantly higher in older age groups (12 and 24 months old; fu = 0.26–0.30) than in younger animals (14, 20, and 40 days old; fu = 0.16–0.18). Binding was best described by a model incorporating a saturable and a nonsaturable binding site. Binding affinity at the saturable binding site was lowest at the extremes of age. Changes in binding at either the saturable or the nonsaturable site were not predicted by changes in TP or FFA with age. Changes in nonsaturable binding were marginally associated with age (p = 0.0952). A 3-fold increase in FFA concentrations was necessary to produce a 1.5-fold increase in VPA fu. There was less than a 2-fold difference in FFA concentrations between the age groups (range 0.219–0.379 mmol/L). Thus, the difference in FFA concentrations between the age groups may not have been large enough to cause measurable differences in displacement of VPA from binding sites. Changes in protein binding may contribute to age-related changes in disposition of VPA observed in the rat. Changes in the serum concentrations of specific FFA or proteins may play a role in the altered VPA binding with age, but changes in total FFA or protein concentrations do not account for the age-related differences observed. Further investigation is required to identify the mechanism(s) responsible for age-related changes in binding of VPA to serum proteins measured in vitro.