Lysosomal storage disorders (LSDs) are caused by defective enzyme activities in lysosomes, characterized by the accumulation of sphingolipids, glycolipids, oligosaccharides, mucopolysaccharides, the oxidation products of cholesterol, and other biological substances. A growing number of clinical studies have suggested the enhanced efficacy of existing therapies, including enzyme replacement therapy, which is effective when it is initiated during the presymptomatic period. Thus, the identification of disease-affected individuals by newborn screening has been considered an effective platform. Previous studies have suggested that the discrimination of infantile-onset Pompe disease (IOPD) requires multi-step examination of GAA enzyme activity using the fluorometric technique. In sharp contrast, the MS/MS-based technique can identify the population of IOPD and the pseudodeficiency alleles of the GAA enzyme [Liao HC et al. Clin Chem (2017) in press; doi: http://dx.doi.org/10.1373/clinchem.2016.269027]. To determine whether MS/MS-based assay can identify these two populations in Japanese neonates, we first performed a validation study of this assay using flow-injection analysis (FIA)-MS/MS and liquid chromatography (LC)-MS/MS followed by examination of GAA enzyme activity in our population. By minimizing the effect of substrate-derived in-source decomposition products, the activities of 6 LSD enzymes were quantified in FIA-MS/MS and LC-MS/MS. The mean value of GAA activity with IOPD, pseudodeficiency alleles, and healthy controls by FIA-MS/MS were 1.0 ± 0.3 μmol/h/L (max, 1.3; min, 0.7; median, 1.2; n = 3), 2.7 ± 0.7 μmol/h/L (max, 4.5; min, 1.5; median, 2.5; n = 19), and 12.9 ± 5.4 μmol/h/L (max, 29.6; min, 2.5; median, 11.0; n = 83), respectively. These results suggest that the population of GAA with pseudodeficiency alleles has approximately 20% of GAA enzyme activity compared to controls, providing the preliminary evidence to estimate the cut-off values in the Japanese population using this technique.