The sensitivity of amino acid analysis by electrospray-tandem mass spectrometry (ESI-TMS) in differential diagnosis of the different forms of hyperphenylalaniemias (HPA) was studied in a total number of 78 dried blood samples from neonates with increased phenylalanine (Phe) concentrations (> 150 micromol/l). Simultaneous quantitative analysis of Phe and tyrosine (Tyr) were performed by ESI-TMS. By means of the Phe/Tyr-ratio it was possible to differentiate between phenylketonuria (PKU; n = 23) and nonPKU-HPA patients (n = 35; P < 0.001) with a sensitivity of 100%. Neonates with transient HPA (n = 9) showed a lower Phe/Tyr-ratio compared to the nonPKU-HPA patients (P < 0.001). Amino acid analysis by ESI-TMS represents a powerful diagnostic tool in differential diagnosis of HPA and is an appropriate technique in the setting of neonatal screening based on dried blood spots.