Improving early detection, diagnosis, treatment monitoring and prognosis of cancer will require rapid and high throughput detection, identification, and measurement of multiple biomarkers. In this study, we demonstrate the versatility of the innovative SELDI ProteinChip(R) MS technology for the rapid, reproducible and simultaneous identification of four well-characterized prostate cancer-associated (PCA) biomarkers, prostate specific antigen (free and complexed forms), prostate specific peptide, prostate acid phophatase and prostate specific membrane antigen in cell lysates, serum and seminal plasma. Proteins corresponding to the mass of these biomarkers could readily be captured and detected using either chemically defined or antibody coated ProteinChip(R) arrays. Several (yet to be identified) proteins were found upregulated in cell lysates of pure populations of PCA cells procured by laser capture microdissection (LCM) when compared with mass spectra of normal cell lysates. Coupling LCM with SELDI provides tremendous opportunities to discover and identify the signature proteins associated with each stage of tumor development. Collectively, these observations demonstrate the potential of SELDI for the discovery and simultaneous detection of and clinical assay development for PCA biomarkers in complex biological mixtures.