GSD type II is a progressive lethal metabolic myopathy, and GAA activity can be accurately detected by muscle tissue, skin fibroblasts, and peripheral blood lymphocytes, but all have limitations. activity assay takes at least several weeks; poor purity of lymphocyte isolation will lead to false negatives of the assay. The determination of GAA activity by DBS and leukocyte assay under acarbose inhibitor is noninvasive, accurate and rapid, which is important for early diagnosis of GSD type II patients. The DBS method established in this study for the determination of GAA activity had intra-batch variation within 8% and inter-batch variation within 10%, which shows that the method has good precision. The DBS method is convenient for blood collection and storage and transportation, which provides great convenience and feasibility for GAA determination in the field. Moreover, the DBS method can better meet the requirements of neonatal disease screening in terms of method precision, specimen storage conditions, and its high-throughput assay.The incidence of GSD type II varies greatly with population and geographic distribution [1,3]. Due to the non-specific clinical presentation of the disease and the limited awareness of clinicians in China, only 31 cases of GSD Ⅱ have been reported so far in mainland China [10-14], and the epidemiological data on the incidence of GSD Ⅱ in mainland China have yet to be further clarified after the screening of newborns for the disease. In this study, we established that the median GAA activity in the normal neonatal and child-adult groups by the DBS method was 27.09 and 16.36 pmol/(punch?h), respectively, and was significantly higher in the neonatal group than in the child-adult group.The median values of 46.8 and 45.7 pmol/(punch?h) in the Australian and Danish normal neonatal groups by Meikle et al [14] using the quantitative immunoassay /(punch?h), while the median for the Australian adult group was 24.4 pmol/(punch?h), and Zhang et al [7] reported a median GAA of 20 pmol/(punch?h) for the normal adult DBS method, similar to our findings. Since the GAA enzyme activities of the patients with GSD type II involved in the above-mentioned studies were all below significantly lower than the detection limit, the difference in the normal values between the neonatal and child-adult groups did not affect the diagnosis and screening of patients. In this study, GAA activity was measured in 4 patients with clinically suspected GSD type II to clarify the diagnosis. the DBS method found that the GAA enzyme activities of the patients were all below the detection limit, suggesting that the DBS method can accurately distinguish between patients and heterozygotes. 6 cases of heterozygous enzyme activities crossed the low value area of normal controls, so the DBS method could not distinguish between heterozygotes and normal individuals. The leukocyte method measured the GAA activity in 3 patients and found that the patients had significantly lower activity compared to normal subjects. Combining the clinical symptoms of progressive myasthenia with the findings of cardiac hypertrophy and myocardial biopsy pathology, child 2 was diagnosed as GSD type II infantile, and patients 3 and 4 were diagnosed as GSD type II late-onset. In conclusion, the DBS method for the determination of GAA activity has the advantages of sensitivity, rapidity, high throughput, and ease of sample transport and preservation, and is suitable for the screening and diagnosis of newborns and high-risk groups with GSD type II; the leukocyte method for the determination of GAA activity has the advantages of accuracy, rapidity, and high specificity, and is suitable for the confirmation of diagnosis in suspected patients.