Phenylketonuria is an inherited metabolic disease, which is characterized by increased level of serum phenylalanine (Phe). The quantitative measurement of Phe in the serum is necessary to confirm the disease, and to distinguish phenylketonuria from other forms of hyperphenylalaninemia. In this study, we report a rapid and inexpensive micro-assay for simultaneous detection and quantitative measurement of serum Phe in dry blood-spots. Analysis of the standard curve showed a broad linear Phe range of 120?1800 mumol L?1. Application of this method in conjunction with the standard Guthrie bacterial inhibition assay and high-pressure liquid chromatography in analyzing 34 samples from phenylketonuria patients and control samples produced comparable results, with the regression equation of Y= 0.994X + 0.996. The advantage of this method over the Guthrie bacterial inhibition assay is its ability to measure the serum Phe quantitatively without false positive results. The method was successfully applied to dried blood-spots as well as serum and whole blood samples. The cost per sample is about 20?50 US cents, which is much less than those of high-pressure liquid chromatography and enzymatic commercial kits. The method can be automated, which is suitable for neonatal and mass phenylketonuria screening, especially in developing countries, where funding is a limiting factor.