We report an innovative multiplexed liquidchromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS)-based assay for rapidly measuring a large number of disease specific protein biomarkers in human serum. Furthermore, this approach uses stable isotope dilution methodology to reliably quantify candidate protein biomarkers. Human serum was diluted using a stable isotope labeled proteome (SILAP) standard prepared from the secretome of pancreatic cell lines, subjected to immunoaffinity removal of the most highly abundant proteins, trypsin digested, and analyzed by LC-MRM/MS. The method was found to be precise, linear, and specific for the relative quantification of 72 proteins when analyte response was normalized to the relevant internal standard (IS) from the SILAP. The method made it possible to determine statistically different concentrations for three proteins (cystatin M, IGF binding protein 7, and villin 2) in control and pancreatic cancer patient samples. This method proves the feasibility of using a SILAP standard in combination with stable isotope dilution LC-MRM/MS analysis of tryptic peptides to compare changes in the concentration of candidate protein biomarkers in human serum.