Pancreatic ductal adenocarcinoma (PDAC) is the fourth most frequent cause of cancer mortality in the United States. Because CA 19-9 increases not only in PDAC, but also in benign conditions, there is urgent need for an additional PDAC biomarker. Isotope tags for relative and absolute quantification (iTRAQ) were performed using 6 pairs of PDAC and normal tissues from the same patients, to obtain preliminary PDAC-specific proteins; and verification was performed by multiple reactions monitoring (MRM), using 30 PDAC and 20 normal serum, targeting high-abundant serum proteins without any pre-preparation. As a result, 17 candidate proteins from tissue iTRAQ were verified as potential markers (AUC values > 0.7). Multivariate analysis (MA) demonstrated that a 6-marker panel, consisting of alpha-1 antitrypsin, haptoglobin beta chain, hemopexin, transferrin, zinc alpha-2 glycoprotein, and apolipoprotein A4 from the MRM result, had comparable discriminatory power versus CA 19-9. Our study demonstrated that a combination of iTRAQ on PDAC tissue and verification MRM-MA on individual serum was an efficient method for the development of PDAC multimarkers.