Abstract Protein kinase play pivotal regulatory roles in most cell communication and metabolic pathways. Inhibitors of protein kinases not only hold great promise as therapeutic agents for many diseases, especially cancer, but are also of profound utility in the characterization of signaling pathways because of the centrality of protein phosphorylation as a regulatory process. Therefore, sensitive and widely applicable detection of protein kinase activity will provide a valuable tool to screen protein kinase inhibitors in drug discovery. We previously reported that self-assembled poly-ion complexes (PICs) containing kemptide, protein kinase A specific peptide motif, recognize the phosphorylation of kemptide by protein kinase A by recovery of fluorescence. In this research we found kinase specific substrate peptide with phage display which an efficient primary screening method to detect protein-protein interaction because of its relatively easy, rapid and massive recovery and analysis. Kinase substrate peptide library was made with phage and then library is displayed on capsid protein of phage. After kinase reaction, the phage clones are selected, recovered, and amplified for next round of bio-panning. The candidate clones are selected according for at least 4th round of bio-panning and the resultant peptides are analyzed for specificity. To prepare PICs, found peptide was conjugated with polyehtyleneimine (PEI) labeled with cyanine 5.5 (cy5.5). PICs consisting of peptide and cy5.5 chemically conjugated PEI and poly-L-aspartic acid showed significant fluorescent signal recovery in presence of protein kinase while they hardly showed fluorescent signal in absence of protein kinase. In addition, fluorescent signal did not recover with protein kinase inhibitor even though protein kinase was present. We tried to apply highthroughput screening to perform chemical library screening with 384-well plate. While wells without protein kinase showed strong fluorescent signal, wells containing protein kinase inhibitors represented no fluorescent signal, which is fully consistent with previous results and our expects. We conclude that this system is applied to highthroughput screening systems targeting protein kinase inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 343.