The confirmation of type III glycogen storage disease diagnosis is based on histological explorations on to live and/or muscle biopsies that induce some problems of delay and sensitivity. The purpose of this study was to evaluate a fluorimetric technique for the determination of amylo-1,6-glucosidase activity in leukocytes, in order to confirm the diagnosis of type III glycogen storage disease. The method consists in measuring the glucose released by hydrolysis of phosphorylase dextrin limit in the presence of cellular extracts, in 50 volunteers and 18 patients suspected of glycogenosis. Benefits of this technique are linearity, precision (CV = 1.68%), exactitude (CI = 0.17%), its high sensitivity (Sn = 100%) and its specificity (Sp = 96.1%). The phosphorylation of dextrin limit test allows measurement of both transferasic (α-1,4) and hydrolytic (α-1,6) enzyme activities. In conclusion, this non-invasive, and inexpensive assay, can be applied to most of the clinical biology laboratories. Comparison with radiometry and immunoblot indicate a noticeable discriminating capacity between normal subjects, patients with type I and VI glycogenosis, and patients' subgroups of type III glycogenosis.