The isolation and proteomics characterization of Extracellular Vesicles (EVs) from body fluids represents a stimulating challenge to improve our knowledge of biology, considering the vast heterogeneity in terms of phenotype in vivo. Within a complex and dynamic scenario of EVs isolation and characterization protocols, we have recently demonstrated how the Fluorescence-activated Cell Sorting (FACS) strategy can be adopted to isolate total EVs from untouched body fluids enabling proteomics characterization. Here, we reported for the first time the proteomics characterization of EVs single-phenotype by sorting labelled-recognition events related to Leucocyte-derived EVs (Leuko EVs) by combining lipophilic dye/phalloidin and anti-CD45+ antibodies from peripheral blood and tears of enrolled volunteers. More than 90% of proteins identified in both biological fluids were mainly referred to leukocyte phenotype. This strategy was applied to carry out a label-free proteomics on Leuko EVs from tears of Multiple Sclerosis (MuS) patients, demonstrating as they are enriched with an encoded protein cargo, reflecting the neuroinflammatory condition of MuS (TGFB1 and NFE2L2, upstream regulators activated) and the endothelial cell proliferation together with an increase of vascular networks (AGNPT2 and VEGF, upstream regulators activated) which in turn reflect pro-angiogenic processes predicted as significantly up-regulated downstream effects. Our data showed that FACS-Proteomics strategy for EVs single phenotype characterization could have groundbreaking avenue for biomarker discovery, exalting the clinical value of tears EVs and helping in a better understanding of the EV-mediated processes in vivo.