AbstractProtein phosphorylation plays a major role in most cell‐signaling pathways in all eukaryotic cells. Disruptions in phosphorylation‐mediated cell‐signaling events are associated with various diseases, including cancer. Here, we applied a fully non‐gel‐based methodology to obtain an initial panel of phosphoproteins from the LNCaP human prostate cancer cell line. The analytical strategy involved enrichment of phosphopeptides by immobilized metal ion affinity chromatography, the use of POROS Oligo R3 to capture phosphopeptides that were not retained with a C18 packing, and gas‐phase fractionation in the m/z dimension to extend the dynamic range of the LC‐MS/MS analysis. In this pilot investigation, 137 phosphorylation sites in 81 phosphoproteins were identified. The characterized phosphoproteins include kinases, co‐regulators of steroid receptors, and a number of cancer‐related proteins.