The ISOLAB NCS phenylalanine determination kit has not been widely applied for neonatal screening and patient follow up in Europe until now. This method, based on fluorescence enhancement of a phenylalanine-ninhydrin reaction product by the dipeptide L-leucyl-L-alanine, was compared with three other procedures: (1) The Quantase kit (Shield Diagnostics) for enzymatic determination of phenylalanine, (2) the standard amino acid analysis by means of ion exchange chromatography, and (3) the Guthrie Test as a bacterial inhibition assay (BIA). Only authentic samples from PKU patients were evaluated: once with the NCS kit and at least once with one of the three other methods. There was good agreement between the results obtained by the NCS kit using dried blood specimens and either of the other three methods, as well as between the NCS kit using plasma samples and the Quantase kit and ion exchange chromatography. Plasma sample measurement by NCS proved advantageous because of the option of measuring each microtiter plate twice by resetting the calibrators, i.e. special standards for plasma samples could be used on the same plate. We conclude that this method should prove time saving and cost effective when both neonatal screening and patient follow up are carried out in the same laboratory.