West Nile virus (WNV) causes severe neurological disease and fatalities in both human and animal hosts. There is currently no effective vaccine or antiviral drug to protect against WNV infection. The West Nile virus NS3 protease, essential for the proteolytic processing of the viral polyprotein precursor, is a promising target for the development of anti-WNV drugs. The SensoLyte® 440 West Nile Virus Protease Assay Kit provides a convenient homogenous assay for high throughput screening of WNV protease inhibitors and for continuous assay of NS3 protease activity using a fluorogenic Pyr-RTKR-AMC peptide. Upon NS3 protease cleavage, Pyr-RTKR-AMC generates the AMC fluorophore that has bright blue fluorescence and can be detected at excitation/emission=354 nm/442 nm.