Mucopolysaccharidoses (MPSs) are characterized by deficient activity of lysosomal hydrolase enzymes, leading to progressive accumulation of glycosaminoglycans. These glycosaminoglycans can be assayed in biofluids as potential markers of disease severity and response to disease-modifying therapies. This study sought to calculate control reference intervals in a largely pediatric population for key MPS biomarkers: heparan sulfate (HS) and dermatan sulfate (DS) in cerebrospinal fluid (CSF) and urine, and CSF monosialic gangliosides GM2 and GM3. We also explored the effect of age on biomarker levels. Biomarker levels were measured using liquid chromatography-tandem mass spectrometry in CSF and urine samples from pediatric and young adult donors and were compared with baseline CSF and urine biomarker levels from an ongoing Phase 1/2 study of children with MPS II. Age-specific reference intervals were estimated for CSF HS, DS, and GM2, and for urine HS, DS, and the sum of HS and DS, after observing that levels of these markers decreased with age. CSF GM3 levels were not found to be age dependent, therefore a single reference interval was estimated for the reference population. In patients with MPS II, levels of HS and DS, respectively, were 6- and 7-fold higher in CSF, and 13- and 30-fold higher in urine than the upper reference interval limits. Establishing age-specific reference intervals will help to optimize biomarker use in clinical studies.