Background: Human milk (HM) is the earliest form of extrauterine communication between mother and infant. We recently showed a functional characterization of HM, unmasking the molecular mechanisms related to EV signaling and its functional role in prematurity. In that study, we identified the need to establish and optimize a standard isolation protocol for human milk extracellular vesicles (mEVs). Results: ExoGAG and UC proved to be the most efficient of the four techniques compared for mEVS isolation. However, ExoGAG compared to UC provided a higher concentration of total and vesicle-related proteins and peptides and a higher glycoprotein count keeping all the glycan subgroups. Despite ExoGAG and UC show similar vesicle profiles in terms of size, concentration, tetraspanin subpopulations and EV markers, ExoGAG was the most efficient technique in terms of accuracy, consistency and reproducibility for omics studies. Furthermore, results allowed us to identify that EVs are involved in the signaling pathways of infant biological development, immune system maturation and protein metabolism. Methods: Four mEVs isolation methods were compared: ultracentrifugation (UC), size exclusion chromatography (SEC), immunoprecipitation with tetraspanin CD9 (IP_CD9) and ExoGAG. Three pools of human milk (each composed of samples from ten donor mothers) were used and isolation of mEVs was performed starting from the same volume for each method. The proteomic, transcriptomic and glycomic composition of the extracellular vesicles obtained after isolation was then analyzed for each method. The sensitivity, specificity and quality of the results were also determined. A comparison of the results obtained common to all methods was carried out in order to establish the possible signaling pathways related to mEVs. Conclusions: This study establishes UC and ExoGAG as reliable methods for mEVs isolation and describes its protocol, being ExoGAG the most efficient. Also, confirms that mEVs play a role in the defense system against external agents (specific role in the immune system pathway) and in the correct establishment of the neural structure (developmental pathway), while providing all the nutritional requirements for the correct growth of the newborn (metabolic pathway).