Accordingly, in this study, we developed an analytical method in our laboratory for simultaneous pretreatment and better quantification of the activities of five enzymes important in MPS—α-iduronidase (IDUA, EC 3.2.1.76) in MPS I, iduronate-2-sulfatase (I2S, EC 3.1.6.13) in MPS II, N-acetyl-α-glucosaminidase (NAGLU, EC 3.2.1.50) in MPS IIIB, N-acetylgalactosamine-6-sulfatase (GALNS, EC 3.1.6.4) in MPS IVA, and N-acetylgalactosamine-4-sulfatase (ARSB, EC 3.1.6.12) in MPS VI—by using LC-MS/MS. Here, GALNS is linked to mucopolysaccharidosis type 2.