Traditional AA dose-response studies utilize many animals for evaluation of growth performance, and it is hypothesized that a new experimental design based on modem analytical techniques can reduce the number of used animals. The objective was to evaluate a short-term approach with a low number of pigs based on plasma metabolites as a method to determine the dietary Ile, Leu, and Val requirements. Three separate 6 x 6 Latin square experiments having 6 replicates per treatment were conducted with 6 diets containing increasing concentrations of Ile, Leu, and Val which were fed to 6 pigs (BW 8-9 kg) for 2 days, each without a wash-out period for a period of 12 days. The diets were prepared and used in 3 previous traditional-design dose-response studies and had been stored at 20 degrees C. Blood samples were collected at the end of each 2-day period, and plasma was analyzed for AA and other metabolites using a metabolomics approach. Out of the 18 analyzed plasma AA, 11, 16, and 3 AA for Ile, Leu, or Val, respectively, showed linear or quadratic responses (P < 0.05) which could be linked to animal growth. The same was found for 4 non-AA metabolites in the Ile, and for 7 non-AA metabolites in the Leu study. 3-Methyl-2-oxovaleric acid, ketohexanoic acid, and a-ketoisovaleric acid were discriminating metabolites for both Ile and Leu. It was possible to fit least squares means of 5, 14, and 2 metabolites in the Ile, Leu, and Val experiments to curvilinear-plateau, broken-line, or quadratic models and thereby estimate an optimum dietary BCAA level. The average optimum BCAA levels across metabolites and models were 0.54 standardized ileal digestible (SID) Ile:Lys, 1.04 SID Leu:Lys, and 0.68 SID Val:Lys which were close to optimums of 0.52, 0.93, and 0.70 found in the previous dose response studies based on animal growth performance. In conclusion, certain plasma metabolites could be used to estimate Ile, Leu, and Val requirements, and 2 days of adaptation to a new diet was sufficient to reflect relevant biological changes in the blood to different levels of dietary AA in the current study.