Multiple reaction monitoring (MRM) is a mass spectrometry technique based on selection of a peptide ion and one or more characteristic fragment ions. The MRM method has previously been shown to be specific, accurate, and reproducible between laboratories. Through the use of retention time windows, MRM-based quantitation can also be multiplexed to analyze and quantitate hundreds of proteins per run, thus increasing the throughput of this type of assay and making it rapid enough for clinical applications. Although MRM-based quantitation is typically used for the clinical validation of potential biomarkers, in this paper, we demonstrate the use of a panel of MRM assays for biomarker discovery. We previously reported on a method for the quantitative analysis of the 45 most abundant plasma proteins, based on liquid chromatography MRM/mass spectrometry, with quantitation accuracy provided by the use of stable isotopically labeled standard peptides. In this paper, we describe the use of this MRM method for the discovery of biomarkers characteristic of the presence or absence of coronary artery disease. Quantitative analysis of peptides specific for 44 of these abundant proteins was performed on plasma from a group of 38 patients classified as coronary artery diseasepositive or coronary artery disease-negative. Statistical analysis of the results resulted in the discovery of a panel of 5 proteins with discriminative concentrations between patients with and without coronary artery disease, with a P value of <0.05. An initial evaluation of the analytic pipeline by cross-validation resulted in an estimated sensitivity and specificity of 74% for the test samples. This demonstrates that MRM-mass spectrometry-based quantitation offers a potential methodology for both biomarker discovery and biomarker validation. Similar to other discovery methodologies (eg, isobaric tags for relative and absolute quantitation), MRM-mass spectrometry provides the sensitivity and accuracy needed in the discovery phase, but, unlike the others, MRM-mass spectrometry offers the high throughput needed for clinical validation. Thus, this advantage eliminates the need for the development and validation of different discovery and clinical validation methods.