West Nile Virus (WNV) is a worldwide concern to the health of the human population. Severe cases of infection can progress into neurological diseases that are potentially fatal due to the lack of treatment or vaccine. The NS2B-NS3 protease is a potential therapeutic target of WNV because of its' vital role in the viral replication cycle. A drug that can inhibit this protease can serve as a treatment for WNV infections. With the use of a fluorescence resonance energy transfer (FRET) assay, a high throughput screen of a chemical library resulted in the discovery of Zafirlukast as a mixed competitive inhibitor for the WNV NS2B-NS3 protease. As an FDA approved treatment of asthma, Zafirlukast is a promising lead compound for optimization as a NS2B-NS3 protease inhibitor. Through a series of structure-activity relationship studies, the significant structural elements of Zafirlukast as a WNV NS2B-NS3 protease inhibitor were identified as the cyclopentyl carbamate and o-toluenesulfonamide. Additionally, a Zafirlukast analog that replaced the cyclopentyl carbamate with a phenyl carbamate was found to have an increased inhibition of 92% at 60 microM and an IC50 of 22.58 microM. Molecular modeling of the Zafirlukast analog bound to the WNV NS2B-NS3 protease revealed mainly hydrophobic interactions with an allosteric site. The o-toluenesulfonamide seems to be important in blocking the NS2B cofactor from binding to the NS3 protein; therefore, inhibiting proteolytic activity.