Phenylketonuria (PKU) is an inherited metabolic disorder in amino acid metabolism. It is due to the deficiency of the enzyme, phenylalanine hydroxylase. Therefore, the conversion of phenylalanine to tyrosine is deficient, i.e. phenylalanine accumulates in the blood giving a concentration of 10–80 mg/dL compared with <2 mg/dL in normal infants. Among the derivatives of phenylalanine present in urine, the largest amounts are phenylpyruvic acid and phenyl lactic acid. The tests used for screening phenylketonuria are Guthrie’s Bacterial Inhibition Test, Ferric Chloride Test, and alkaptonuria are briefly discussed in this chapter.