Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods, and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the ng/mL range in plasma, and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10ng/mL range with %CV’s from 3-15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity-depleted plasma from a healthy donor. Quantitative MRM assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a LOQ in the 1 -10 ng/mL range, and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/mL range. These levels of assay performance represents up to a 1000-fold improvement compared to direct analysis of proteins in plasma by MS, and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-MRM/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.