Pancreatic ductal adenocarcinoma (PDAC) accounts for 85–90% of all pancreatic tumours. The median survival of all PDAC patients is less than 6 months, and the recent 5-year-survival rate is approximately 8%. One of the most crucial reasons for the poor prognosis is the lack of early diagnostic markers for PDAC. To overcome this and improve the outcomes of patients with PDAC, there is an urgent need to identify highly sensitive and specific markers for early detection. The widely used serum-circulating markers for PDAC, carbohydrate tumour–associated antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA), are not sufficiently accurate to be used as early diagnostic markers. In the present study, we employed the TMT method to generate comparative protein profiles of PDAC cell lines. Furthermore, we compared the serum levels of candidate proteins to evaluate the ability to discriminate between PDAC and healthy controls. Using tandem mass tag labelling and LC-MS/MS, we performed comparative analyses of secreted proteins in culturing media obtained from human PDAC cell lines (MIA PaCa-2, PANC-1, and BxPC-3) to identify serum biomarkers for PDAC. In validation studies, we evaluated the discriminatory power of candidate proteins. Of 27 proteins, 13 proteins were identified with the increase of protein levels among these all three cell lines, and Protein X was selected for further analysis. The sera levels of Protein X were significantly higher in the preoperative PDAC patients than those in the postoperative ones (P<0.001). In future study, we will verify this result with more number of samples, and pursue the usefulness of Protein X as a novel diagnostic marker in PDAC.