Extracellular vesicles (EVs) are becoming promising tools for clinical application, either as sources of disease-specific molecular signatures for the unraveling of disease pathophysiology and establishment of novel biomarkers, or as platforms for cell-free nanotherapy. Yet, an unsolved issue is to define standardized techniques for EV isolation allowing data comparison across laboratories worldwide. Considering the difficulties to find this necessary consensus, it has to be stressed out that the outcome of the downstream analysis might be deeply biased by the isolation method, among other variables. Thus, it is crucial that the researcher is aware of the strengths and weaknesses of each method keeping their intended use in mind, and to sufficiently report the methodology details for the results to be comparable and solid. This review aims to present the most widely used EV isolation methods, from the initial differential ultracentrifugation (dUC) to newest approaches. •A pan-method for EVs’ isolation does not exist.•EVs’ isolation method biases the outcome of downstream analysis.•EV final use will determine the strategy of isolation.•The best EV isolation strategy might be a combination of different methods to reach the ideal purity and recovery.•Correct reporting of the EV isolation method in papers will lead to comparable and solid results.