Mucopolysaccharidosis (MPS) is a genetic disorder characterized by the accumulation of glycosaminoglycans in the body. Of the multiple MPS disease subtypes, several are caused by defects in sulfatases. Specifically, a defect in iduronate-2-sulfatase (ID2S) leads to MPS II, whereas N-acetylgalactosamine-6-sulfatase (GALN) and N-acetylgalactosamine-4-sulfatase (ARSB) defects relate to MPS IVA and MPS VI, respectively. A previous study reported a combined assay for these three disorders in a 96-well plate using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technique (Kumar et al., Clin Chem 2015 61(11):1363-1371). In our study, we applied this methodology to a Japanese population to examine the assay precision and the separation of populations between disease-affected individuals and controls for these three disorders. Within our assay conditions, the coefficient of variation (CV, %) values for an interday assay of ID2S, GALN, and ARSB were 9%, 18%, and 9%, respectively (n = 7). The average enzyme activities of ID2S, GALN, and ARSB in random neonates were 19.6 ± 5.8, 1.7 ± 0.7, and 13.4 ± 5.2 μmol/h/L (mean ± SD, n = 240), respectively. In contrast, the average enzyme activities of ID2S, GALN, and ARSB in disease-affected individuals were 0.5 ± 0.2 (n = 6), 0.3 ± 0.1 (n = 3), and 0.3 (n = 1) μmol/h/L, respectively. The representative analytical range values corresponding to ID2S, GALN, and ARSB were 39, 17, and 168, respectively. These results raise the possibility that the population of disease-affected individuals could be separated from that of healthy individuals using the LC-MS/MS-based technique.