Extracellular vesicles (EVs) represent different types of membranous nanosized vesicles which are produced and released by all cells in the body. Number of studies analysing EVs and their importance in cell to cell communication is continuously growing. Due to EV’s nano size, gold standard imaging method for their visualisation is electron microscopy (EM), both in scanning and transmission mode. In the literature, considerable variability between their preparation protocols and visualisation methods was observed so the main goal of this study was to optimise EV preparation protocol for EM. Results show that conductivity of the hydrophilic polycarbonate membrane can be obtained by gold-palladium (Au-Pd) sputtering of its surface but precaution must be taken when choosing the thickness since it affects hydrophilicity of the membrane and the attachment of the sample. Regarding fixation, resulting images did not show significant difference in EV shape and size when comparing two fixatives, glutaraldehyde and paraformaldehyde, and two fixation times, 1 h at room temperature (1 h/RT) and 24 h at +4 °C (24 h/+4 °C). In contrast, resulting images did show differences in shape and size when comparing critical point dried (CPD) and air dried EVs indicating that CPD preserves surface structure. Lastly, scanning transmission electron microscopy (STEM) images comparing four contrasting solutions, osmium tetroxide aqueous solution, uranyl acetate (UA), UranyLess TM, and UA-ZeroTM, show that they stain different structures. Taken together, findings in this thesis reveal the complexity of EV preparation protocol for EM and indicate that further research is needed for its optimisation, especially regarding fixation and staining.