The efficacy of the tandem mass spectrometry as a tool for the newborn screening of phenylketonuria, an inherited metabolic disorder, was investigated. Precision, reproducibility, selectivity and sensitivity were validated for phenylalanine and tyrosine measurements from dried blood spots. Bland-Altman plots were used to assess the agreement with conventional methods like fluorometry and ion exchange chromatography. The utility of the phenylalanine/tyrosine ratio for discrimination between mild hyperphenylalaninemia and classical types of phenylketonuria was investigated. Depending on concentration levels of phenylalanine and tyrosine the within-run and between-run assay variability ranged between 4.2% and 12.7%. Higher recoveries and a lower detection limit were found for the mass spectrometric method when compared to the fluorometric method. Pearson correlation coefficients of 0.91 for tandem mass spectrometry vs. fluorometric method, as well as 0.95 for tandem mass spectrometry vs. ion exchange chromatography were calculated. The closest agreement between methods was observed between tandem mass spectrometry and ion exchange chromatography. The results demonstrate a high efficacy of the tandem mass spectrometric method for quantitative determination of phenylalanine and tyrosine from dried blood spots. The phenylalanine/tyrosine ratio is crucial to improve the specificity and positive predictive value for the diagnosis of classical phenylketonuria.