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The receptor for advanced glycation end products (RAGE) is a multiligand receptor, as it may bind a variety of pro-inflammatory endogenous, as well as pathogen-derived molecules, including members of S100 protein family, high mobility group box protein-1, amyloid peptides and fibrils, lipopolysaccharide or F-protein from respiratory syncytial virus. RAGE-ligand interactions have been intensively studied in relation with their contribution to pathological alterations in inflammatory disorders, diabetes, neurodegeneration and cancer, and the possibility to be targeted for therapeutic interventions. Of these interactions, S100P-RAGE axis was shown to be involved in pancreatic and colon cancers. Our goal is to find new compounds to block the oncogenic pathways driven by S100P-dependent RAGE activation. In this study we describe two fluorescence-based methods which we set up to measure RAGE-S100P binding and be rapidly and effectively used in high-throughput screening for compounds that can interfere with the binding. One method relies on the specific binding of a EGFP fusion of S100P to soluble RAGE, pre-adsorbed in 96-well plates. The second method employs a time-resolved FRET pair using S100P-EGFP and an antibody conjugated with Terbium chelate directed against the His-tag of the soluble RAGE. We discuss the advantages of each method, their use for competitive binding assays and the results of a natural product library screening which led us to the identification of several molecules that can block RAGE-S100P interaction. The evaluation of the newly found inhibitors in cell culture cancer models is currently undertaken. In conclusion, we designed and developed two reliable, rapid and inexpensive methods to be used for high-throughput screening of compounds, and identified several putative S100P-RAGE inhibitors. [ABSTRACT FROM AUTHOR]