Abstract A simple enzymatic spectrophotometric method has been described for the quantitative determination of phenylalanine in serum. The method is based upon the measurement of the absorption of the enol-borate complex of phenylpyruvic acid generated enzymatically from phenylalanine by l-amino acid oxidase of snake venom. For elevated serum levels of phenylalanine the method is rapid, specific, and precise. It should be useful as a confirmatory test in the diagnosis of suspected phenylketonuria and in the evaluation of the effectiveness of a diet low in phenylalanine. The values obtained by this method agree well with those in the literature obtained by other techniques. Tyrosine and tryptophan are also determined by this method, and a suitable modification of the method is described which should allow an accurate estimation of these amino acids and phenylalanine in normal serum.