The dynamics of biological systems depend on complex cell-cell interactions. Extracellular vesicles (EVs) are nanosized vehicles of intercellular communication which shuttle several molecules from cell to cell, thus influencing relevant biological processes. Previous literature based on EV isolation from cell culture medium highlighted how EVs in the peripheral nervous system could be important mediators in the molecular dialogue between neurons and Schwann cells. Here, we aim to extract EVs directly from peripheral nerve tissue, as this would give the unique opportunity to characterize EVs from the biologically complex nerve context. We propose a possible strategy for nerve-derived EV isolation and characterization from mouse peripheral nerves and we describe the workflow from tissue dissociation to EV enrichment with steps of filtration and ultracentrifugation, inspired by published protocols for EV isolation from other tissue types. While considering potential risks and pitfalls of tissue-derived EV studies, our preliminary data suggest that we possibly isolated genuine nerve-derived EVs. Electron microscopy investigations showed presence of vesicular-like structures, further characterized by means of nanoparticle tracking analysis for sizing and concentration and Western blotting for marker detection. Even though a more extensive characterization of nerve-derived EVs as well as protocol optimizations are needed, we believe that the study of these nerve-derived EVs could open the possibility to investigate their relevance in healthy and pathological states, especially in the context of nerve regeneration after damage.