A simple and rapid method for the determination of L-phenylalanine using Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating) has been developed. This method involves the oxidative deamination of L-phenylalanine using L-phenylalanine oxidase from Pseudomonas sp. P-501, and the colorimetric determination of hydrogen peroxide formed from L-phenylalanine with 4-aminoantipyrine and N,N'-dimethylaniline in the presence of peroxidase. This method requires a smaller quantity of sample and is less time-consuming than those previously reported. The method can be used for the direct assay of serum L-phenylalanine levels without pretreatment.