Phenylketonuria (PKU) is a disorder characterized by an interruption in the conversion of phenylalanine to tyrosine, a reaction catalyzed by phenylalanine hydroxylase (PAH). Animal models of PKU used in this study were induced by daily subcutaneous injections of pups with alpha-methylphenylalanine plus phenylalanine in utero and postnatally from day 4 to day 14. Dry blood and plasma were utilized to measure phenylalanine concentration in PKU rats. The results indicated that the concentration of phenylalanine is higher and more stable in plasma than dry blood. Precolumn derivatization of dried blood and plasma free amino acids were conducted with phenylisothiocyanate (PITC). The phenylthiocarbamyl (PTC) derivatives were separated on a reversed-phase C-18 column (15 cm x 4.6 mm). A gradient high-performance liquid chromatography method with two eluents, 0.1 M sodium acetate buffer and 100% acetonitrile was developed to facilitate the separation of nine amino acids within 11 min. Tyrosine and phenylalanine eluted the column at 5.4 and 9.4 min, respectively. This method provides a quick and reliable technique for neonatal screening.