Early detection of prostate cancer and determination of its aggressiveness are critical factors that influence treatment outcomes. To aid in the clinical decision making, novel biomarkers are being sought. Direct, global-scale examination of primary human specimens provides the most relevant picture of the tumor machinery and its perturbations, and this information is highly significant in the context of biomarker discovery. In the pilot study reported here, we focused on mapping of the phosphoproteome in human prostate cancer specimens obtained from a tissue repository. A gel-free proteomic strategy included whole proteome digestion, phosphopeptide enrichment with immobilized metal ion affinity chromatography (IMAC), and phosphoprotein identification via LC-MS/MS and database searches. We applied this strategy to obtain phosphoprotein signatures from a set of five specimens. Phosphoproteins were characterized from each specimen. The phosphoprotein panels included 16-23 phosphoproteins that encompassed 18-30 phosphorylation sites. Some of proteins/sites were characterized in multiple specimens, whereas the majority of sites were found in single specimens. The characterized panels include caldesmone, desmin, HSP beta-1, synaptopodin-2, filamin-C, tensin-1, and others. In summary, the study showed that cancer-relevant phosphoproteins can be characterized directly from archived prostate tumor specimens, establishing the groundwork for further biomarker discovery.