The maturation of mass spectrometry technologies has provided a rich opportunity to interrogate protein expression patterns in normal and disease states by applying expression protein profiling methods. Major goals of this research strategy include the identification of protein biomarkers that demarcate normal and disease populations, and the identification of therapeutic biomarkers for the treatment of diseases such as cancer [1]. Prostate cancer is one disease that would greatly benefit from implementing mass spectrometry based expression profiling methods because of the need to stratify the disease based on molecular markers. In this review article will summarize the current mass spectrometry based methods to identify and validate biomarkers in human prostate cancer. Lastly, we propose a reverse proteomic approach implementing a quantitative mass spectrometry research strategy to identify and quantify biomarkers implicated in prostate cancer development. With this approach, the absolute levels of prostate cancer biomarkers will be identified and quantified in normal and disease samples by measuring the levels of native peptide biomarkers in relation to a chemically identical, but isotopically labeled reference peptide. Ultimately, a centralized prostate cancer peptide biomarker expression database could function as a repository for the identification, and validation of protein biomarker(s) during prostate cancer progression