In our study we have developed an HPLC-ESI-MS/MS method for qualitative and quantitative analysis of underivatized amino acids on dry blood spots. The sensitive and specific instrumental performances permitted the chromatographic separation of 40 amino acids and their isomers within 10 min. The method has been set up for cases of suspected metabolic diseases revealed by newborn screening. What is new is that it is applied on the same blood spots used for newborn screening, instead of plasma, in order to avoid involvement of doctors, increased anxiety for parents, stress for patients for plasma collection, long time of waiting and further costs for analysis.