Lipopolysaccharides (LPS), otherwise termed endotoxins, are an integral part of the outer leaflet of the outer-membrane of Gram-negative bacteria. Lipopolysaccharides play a pivotal role in the pathogenesis of Septic Shock, a major cause of mortality in the critically ill patient, worldwide. The sequestration of circulatory endotoxin may be a viable therapeutic strategy for the prophylaxis and treatment of Gram-negative sepsis. We have earlier shown that the pharmacophore necessary for small molecules to bind LPS is simple, comprising of two protonatable cationic functions separated by about 15 Å, permitting the simultaneous interaction with the negatively charged phosphates on lipid A, the toxically active center of endotoxin. In this report, we employ high-throughput screening methods, using a novel fluorescent probe displacement method. Searches in three-dimensional structure databases yielded about ∼ 4000 commercially available small molecules, each possessing two cationic functions spaced approximately 15 Å apart. Approximately 400 such compounds have been screened in an effort to validate the method by which high-affinity endotoxin binders can be identified. We show that the IC50 values that are obtained from the fluorescence-based primary screen are correlated both to the enthalpy of binding, as measured by isothermal titration calorimetry, as well as to biological potency in vitro assays. By performing rapid toxicity screens in tandem with the bioassays, lead compounds of interest can be easily identified for further systematic structural modifications and SAR studies.