The Northeastern region of Thailand is well known to have a high incidence and mortality of cholangiocarcinoma. Protein phosphorylation status has been reported to reflect a key determinant of cellular physiology, but identification of phosphoproteins can be a problem due to the presence of phosphatase. Growing evidence indicates that exosomes are stable towards circulating proteases and other enzymes in human blood and can be recognized before the onset of cancer progression.Here an in vitrometastatic model of isogenic cholangiocarcinoma cells was used to provide insight into the phosphorylation levels of exosomal proteins derived from highly invasive cells. Gel-based and gel-free proteomics approaches were used to reveal the proteins differentially phosphorylated in relation to tumor cell phenotypes. Forty-three phosphoproteins were identified with a significant change in phosphorylation level. Phos-tag western blotting and Immunohistochemistry staining was then employed to validate the candidate phosphoproteins. Heat shock protein 90 was successfully confirmed as being differentially phosphorylated in relation to tumor malignancy. Importantly, the aberrant phosphorylation of exosomal proteins might serve as a promising tool for the development of a biomarker for metastatic cholangiocarcinoma.