Glycogen storage disease type Ib (GSD1b) and G6PC3-deficiency are rare autosomal recessive diseases caused by inactivating mutations in SLC37A4 (coding for G6PT) and G6PC3 , respectively. Both diseases are characterized by neutropenia and neutrophil dysfunction due to the intracellular accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), a potent inhibitor of hexokinases. We recently showed that the use of SGLT2 inhibitor therapy to reduce tubular reabsorption of its precursor, 1,5-anhydroglucitol (1,5-AG), a glucose analog present in blood, successfully restored the neutropenia and neutrophil function in G6PC3-deficient and GSD1b patients. The intra-individual variability of response to the treatment and the need to adjust the dose during treatment, especially in pediatric populations, can only be efficiently optimized if the concentration of 1,5-AG in blood is monitored during treatment, together with the patients' clinical signs and symptoms. Monitoring the 1,5-AG levels would be greatly simplified if it could be performed on dry blood spots (DBS) which are easy to collect, store and transport. The challenge is to know if a suitable method can be developed to perform accurate and reproducible assays for 1,5-AG using DBS. Here, we describe and validate an assay that quantifies 1,5-AG in DBS using isotopic dilution quantitation by LC-MS/MS that should greatly facilitate patients' follow-up. 1,5-AG levels measured in plasma and DBS give comparable values. This assay was used to monitor the levels of 1,5-AG in DBS from 3 G6PC3-deficient and 6 GSD1b patients during treatment with SGLT2 inhibitors. We recommend this approach to verify the adequate therapeutical response and compliance to the treatment in G6PC3-deficient and GSD1b patients treated with SGLT2 inhibitors. • Neutropenia and neutrophil dysfunction in G6PC3-deficientcy and GSD1b is successfully treated by SGLT2 inhibitor therapy. • SGLT2 inhibitors reduce circulating 1,5-anhydroglucitol (1,5-AG) concentration through reduction of tubular reabsorption. • 1,5-AG monitoring in blood is recommended to verify adequate therapeutical response and compliance to SGLT2 inhibitors. • We developed and validated a reliableLC-MS/MS assay to quantify 1,5-AG in DBS that should facilitate patient follow-up. [ABSTRACT FROM AUTHOR]