Background: Tetrahydrobiopterin is a coenzyme of phenylalanine hydroxylase and other enzymes essential for the synthesis of tyrosine and other neurotransmitters. Despite proper nutritional regulation of blood phenylalanine levels, tetrahydrobiopterin deficiency results in progressive neurologic illness. Objective: The study aims to optimize a mass spec-dependent assay for quantitative measurement of biopterin and neopterin as distinctive markers of the atypical phenylketonuria due to tetrahydrobiopterin deficiency. Patients and methods: The study enrolled 46 patients with typical hyperphenylalaninaemia, 14 atypical cases, and 50 healthy children as a control group. Quantitative measurements of biopterin, neopterin, phenylalanine, and tyrosine were performed by ultra-performance liquid chromatography-mass spectrometry in the dried blood spots of patients and control samples. Results: Validation of the analytical protocol was performed at a biopterin and neopterin concentration range from 0 to 100 nmol/l. The regression coefficients for the linearity of the calibration curves exceeded 0.98. The lower limit of detection of biopterin and neopterin ranged from 1.5 to 2.5 nmol/l. The intra-day and inter-day precision and accuracy ranged from 96% - 105%, and from 97% - 110%. The mean recoveries were 105 ± 7% for biopterin and 106 ± 9% for neopterin. The short and long-term stability of the stored samples was seven days at room temperature and 12 weeks at -20 °C. Biopterin and neopterin were significantly higher in the classic group compared to the atypical and control group (p < 0.05), with their level being in the order classic > control > atypical group. Phenylalanine levels had a significant positive correlation with biopterin and neopterin levels in classic phenylketonuria (p < 0.05). Conclusions: The performance of the developed assay for biopterin and neopterin in the dried blood spot by ultra-performance liquid chromatography-mass spectrometry was accurate and precise thus, provide a legitimate diagnostic tool for cases of atypical phenylketonuria.