Extracellular vesicles (EVs) and cell-derived vesicles (CDVs), generated by fragmenting cellular membranes, have both been explored as therapeutic delivery vehicles. Surface proteins on these vesicles are of great importance as they are characteristic to the cell of origin and modulate vesicle interactions with target cells. Here, we introduced a high-throughput fluorescence correlation spectroscopy (ht-FCS) approach capable of characterizing vesicle surface proteins across a large number of samples. We used automated screening and acquisition of FCS data to profile surface proteins of cell-derived vesicles with high fidelity based on changes in diffusion time upon antibody-vesicle interactions. We characterized vesicles generated from 4 cell types using antibodies for known exosome biomarkers. The ht-FCS technique presented here offers the capability to screen EVs or cell-derived vesicles against a library of surface markers or to screen a library of cell-derived vesicles for a specific identifying marker at a high speed.