In the brain, extracellular glutamate (Glu) levels are maintained low by efficient transporters, whose dysfunction is implicated in neuronal hyperexcitability, excitotoxicity, and many neurological diseases. While many methods can estimate Glu uptake in vitro or ex vivo, a limited number of techniques addresses Glu transport in vivo. Here, we used in vivo microdialysis in a two-in-one approach combining reverse dialysis of isotopic Glu to measure uptake ability and zero-flow (ZF) quantitative microdialysis to sample and quantify extracellular Glu levels. We applied the microdialysis protocols to wild-type mice and DMSXL mice, a transgenic model of human myotonic dystrophy type 1 (DM1) neuromuscular disease, characterized by low levels of the EAAT-2/GLT1 glutamate transporter, among other molecular and functional brain defects. DMSXL mice displayed a ~20% reduction in GLT1 levels in the cortex, and our microdialysis protocol was sensitive enough to unveil in vivo a decrease in cortical Glu uptake. Additionally, on the same animals, we found abnormal extracellular Glu levels determined by ZF method in line with GLT1 defects. However, the “plateau” profile of Glu ZF data was different from that usually obtained with the theoretical ZF model. Three mathematical models were used to fit the shape of ZF Glu data in both wild-type and DMSXL mice. The use of mathematical fitting on the observed “plateau” shape revealed a striking positive correlation between Glu uptake and Glu levels in DM1 mouse strain. In conclusion, we propose a sensitive two-in-one microdialysis approach that is robust enough to reveal significant differences in neurotransmitter uptake and extracellular levels through the analysis of a relatively low number of animals (n≤6 per group).