Maple syrup urine disease (MSUD) is an innate metabolic problem caused by a deficiency of the branched-chain ketoacid dehydrogenase (BCKDH) complex. MSUD is characterized by the accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine, alloisoleucine, valine in urine, blood and spinal fluid and is a rare autosomal disease. For quantitation of branched amino acids in DBS samples LC-MS/MS based methods are useful for rapid confirmatory diagnosis when MSUD patients are diagnosed by clinical findings or family history. Detection and quantification of Allo-isoleucine in DBS samples of potential MSUD cases, in conjunction with the determination of the leucine/phenylalanine ratio, would be a useful approach to shorten the time during the confirmatory diagnostic process.