Updated FTP location. Extracellular vesicles (EVs) including exosomes are secreted into extracellular fluids, which are found in most body fluids. Since EVs contain a number of biologically active molecules such as proteins and mRNAs, there has been increasing interest in EVs as a carrier of potential biomarkers for various diseases. We previously reported that chelating reagent enhanced the recovery rate of EVs from serum, plasma, and urine. In this study, we used bile EVs which packaged biomarker molecules secreted from CCA lesion. Then we performed proteomic analysis of EVs isolated from human bile in CCA and choledocholithiasis (biliary stone) for CCA biomarkers discovery.