Inherited lysosomal storage diseases (LSDs) are rare, and diagnosis is often delayed for 7-10 years. Since the therapies have become available for a limited number of LSDs, (Fabry, Gaucher, Pompe, and MPS-1), early diagnosis of treatable LSDs can be lifesaving or ameliorating and allows timely treatment before irreversible damage occurs. Recently, the use of dried blood spot test (DBS) for newborn screening of LSDs has been proposed for newborn screening tests. They are noninvasive, sensitive, and specific assays with the further advantage of a fast turnaround time compared to measurement in leukocyte and/or fibroblast culture. We aimed to determine the reference intervals for lysosomal enzyme activities of newborn babies in our population and to investigate the effect of gestational week on enzyme activity. One hundred thirty healthy newborn babies (70 girls, 60 boys) were included into the study. alpha-Glycosidase, beta-glycosidase, and alpha-galactosidase activities in DBS samples of newborns were determined fluorometrically. Reference intervals were calculated using Dixon's rule and percentiles of 2.5-97.5. Cutoff limits (5 %) for alpha-glycosidase, beta-glycosidase, and alpha-galactosidase activities were 0.57, 0.92, and 2.18, respectively. alpha-Galactosidase activity was higher in girls compared to boys (p < 0.05). Interestingly, alpha-glycosidase and beta-glycosidase activities of newborns who were delivered before 38 weeks were significantly lower than those who were delivered at 39-40 weeks.