G protein-coupled receptors (GPCR) are the largest family of membrane proteins in the human genome and represent targets of about 30% of the marketed drugs. Among GPCRs, metabotropic glutamate receptors (mGluRs) are interesting targets as they regulate neuronal excitability and synaptic transmission in the central nervous system, and are involved in various brain diseases. Due to the lack of specific tools, their specific subunit composition in native tissues is still difficult to determine. Finding highly specific tools to target each mGluR subunit is still a challenge that single domain antibodies (VHH or nanobodies) could overcome. In this study we developed nanobody-based biosensors specific to mGlu1 and mGlu5 subunits as innovative tools to quantify mGluRs in native tissues. By combining the high affinity and specificity of nanobodies with time-resolved fluorescence resonance energy transfer (TR-FRET) technology, these tools enable proximity detection between subunits of mGluR group I homodimers, mGluR1 and mGluR5. This approach was first set up on HEK293 cells, transiently expressing mGluR1 or mGluR5, to validate the specificity of our biosensors. Preliminary results on different mouse brain regions allowed us to quantify the relative expression at the cell surface of mGluR1 and mGluR5 homodimers. Our results show that mGluR5 is distributed in different brain regions, whereas mGluR1 is mostly expressed in the cerebellum. Overall, this biosensor technology allows to precisely quantify variations of mGluR expression in native tissues. Moreover, nanobodies exhibiting a conformational selectivity on mGluR1 or mGluR5 will be used to determine the endogenous activity of mGluR1 and mGluR5 homodimers.