Reported here is the development of a multiplexed mass spectrometric immunoassay (MSIA) for the detection of myocardial infarction (MI). The assay is the product of a study that systematically progresses from biomarker discovery--to identification and verification--to assay design, data analysis, and statistical challenge. During targeted population proteomics investigations, two novel biomarkers, serum amyloid A1alpha and S-sulfated transthyretin, were found to be responsive to MI. These putative markers were subsequently screened in larger cohorts of individuals to verify their responsiveness toward MI. Upon verification, a multiplexed assay was designed that was capable of simultaneously monitoring the new markers plus a previously established MI-marker (myoglobin). The multiplexed MSIA was applied to two 96-sample sets comprised of 48-MI/48-healthy and 19-MI/77-healthy, which served as training and case cohorts, respectively. Data evaluation using either preset reference levels or multivariate analysis exhibited sensitivities and specificities of >97%. These findings illustrate the importance of using systematic approaches in clinical proteomics to discover biomarkers and produce high-performance assays relevant to disease.