Lysosomal storage disorders (LSDs) are a group of metabolic disorders with various clinical presentations, which complicate diagnosis. A pilot study was performed to test the appropriateness and effectiveness of the newborn screening method for Pompe disease, Fabry disease and mucopolysaccharidosis (MPS) I in dried blood spots using liquid chromatography–tandem mass spectrometry. Around 20 000 newborn samples were analyzed for 3 lysosomal enzyme activities: α-glucosidase (deficient in Pompe disease), α-galactosidase (deficient in Fabry disease) and α-iduronidase (IDUA, deficient in MPS I). Data were used for statistical analysis and to establish sex- and age-dependent reference ranges. Statistically significant higher α-glucosidase, α-galactosidase, and IDUA enzyme activities were observed in female newborns compared to male newborns. Newborns with a higher gestational age have a statistically significant lower α-glucosidase, α-galactosidase, and IDUA enzyme activities compared to newborns with a lower gestational age. For the first time, the data of a large-scale LSD study were used to assess statistical differences in enzyme activity in the newborn population, and these data highlight the importance of using reference intervals for lysosomal enzyme activities in function of sex and gestational age.