Pancreatic cancer has one of the lowest survival rates of all cancers, partly due to the difficulty of detection in its early stages. Recent developments in liquid chromatography – mass spectrometry (LC-MS) technology have made it possible to profile proteins associated with disease in a comprehensive manner. Within this thesis current LC-MS methodology was applied to the problem of pancreatic cancer. A comprehensive list of proteins secreted by the tumor microenvironment was compiled in the first aim. Culture of pancreatic cell lines was used to simulate the tumor microenvironment in vitro and produce a proteome rich in biomarkers. The proteins identified demonstrate the complex signals tumor cells use to communicate with their environment and encompass therapeutic targets and biomarkers. To assay these proteins in serum, a relative quantitative LC-MS method was developed in the second aim. Traditional bioanalytical techniques were incorporated to enhance the utility of the method. Multiple reaction monitoring (MRM)/MS provided sensitivity and specificity to the method and stable isotope dilution (SID) provided correction for losses during sample analysis, enabling precise quantitation and efficient determination of the concentration of over 70 proteins simultaneously. In the third aim, the LC-MS method was used to compare a panel of putative protein biomarkers in pancreatic cancer and control serum. This analysis identified 15 differential proteins with the ability to discriminate pancreatic cancer subjects from their age-matched controls with sensitivity and specificity superior to any other reported pancreatic cancer biomarkers. Future validation of this panel could change the way we diagnose pancreatic cancer.