Anthrax Protective Antigen (PA) has recently been identified as an inhibitor of angiogenesis by binding to its cell surface receptors Capillary Morphogenesis Gene protein 2 (CMG2) and Tumor Endothelial Marker 8 (TEM8). To identify small molecule and peptide inhibitors, we have developed a simple mix-and-measure fluorescence resonance energy transfer (FRET) assay which is suitable for high-throughput screening. We have cloned, expressed, and purified a truncation of the TEM8 extracellular domain. Since this truncation contains 3 native cysteine residues and controlled labeling of this TEM8 construct is critical for FRET assay development, we used site-directed mutagenesis to construct several single cysteine mutants. All mutations were expressed and purified. All mutants also had affinity for PA that was indistinguishable from wild-type. All mutants were labeled with fluorescein maleimide to verify solvent accessibility and labeling efficiency of the cysteine residues. An N-terminal cysteine mutation and one of the native cysteine mutants showed both PA affinity and high labeling efficiency. These mutants were used to optimize the concentration and kinetic parameters of the assay. The assay has been validated in 96-well plates (150 ěl/assay) and gives a high z' using appropriate positive and negative controls. Pilot screening against a panel of FDA approved compounds will begin shortly.