Extracellular glutamate levels are maintained low by efficient transporters, whose dysfunction can cause neuronal hyperexcitability, excitotoxicity, and neurological disease. While many methods estimate glutamate uptake in vitro/ex vivo, a limited number of techniques address glutamate transport in vivo. Here, we used in vivo microdialysis in a two-in-one approach combining reverse dialysis of isotopic glutamate to measure uptake ability and zero-flow (ZF) methods to quantify extracellular glutamate levels. The complementarity of both techniques is discussed on methodological and anatomical basis. We used a transgenic mouse model of human disease, expressing low levels of the EAAT-2/GLT1 glutamate transporter, to validate our approach in a relevant animal model. As expected, isotopic analysis revealed an overall decrease in glutamate uptake, while the ZF method unveiled higher extracellular glutamate levels in these mice. We propose a sensitive and expedite two-in-one microdialysis approach that is sufficiently robust to reveal significant differences in neurotransmitter uptake and extracellular levels through the analysis of a relatively low number of animals.