Prostate cancer is a leading cause of cancer-related death. The current modality of diagnosis, the measurement of serum PSA, not only suffers from lack of specificity, but does not distinguish clinical cases in which current treatment measures would be most successful, i.e. aggressive, life-threatening tumors. A multiplexed MS methodology, selected reaction monitoring-MS/MS coupled with stable isotope dilution (SID), was developed and tested in both cells lines and clinical tissue samples. Standard curves were generated for two peptides representing PSA and one peptide from each of two additional orthogonally validated biomarkers, AMACR and EZH2. The standard curves show high reproducibility, sensitivity, and good linearity. All four peptides were then measured in six clinically relevant cell lines and are in agreement with the biochemical characteristics of each individual cell line. The SID selected reaction monitoring-MS/MS methodology was then transferred to tissue samples, in which the assay shows potential to differentiate benign disease from localized cancer and localized cancer from aggressive metastatic disease. These results establish the preliminary development of a rational targeted MS platform that strives to bridge the gap between discovery and validation of biomarkers for the detection of prostate cancer.