Extracellular vesicles (EVs) are lipid bilayer nanoparticles involved in cell–cell communication, playing roles in both physiological and pathological processes. EVs have recently been isolated directly from solid tissues, which could provide insights into how EVs mediate communication between cells in vivo. Electron microscopy, including three-dimensional (3D) electron tomography, enables visualization of EVs within their native environments, overcoming limitations of traditional bidimensional imaging. This chapter outlines a detailed protocol for visualizing EVs in human tissues using 3D electron tomography, emphasizing the preparation of samples, including fixation, sectioning, and image acquisition. It provides details on the challenges of accurate imaging of EVs through 3D electron tomography and offers insights to improve the resolution and quality of EV visualization in tissue samples. This method allows the visualization of EVs in their native environment, thus preserving their characteristics, which can be altered by the isolation process.