West Nile virus (WNV), a member of the Flaviviridae family, is a mosquito-borne pathogen that causes a great number of human infections each year. Neither vaccines nor antiviral therapies are currently available for human use. In this study, a WNV NS2B–NS3 protease inhibitor with a 9,10-dihydro-3H,4aH-1,3,9,10a-tetraazaphenanthren-4-one scaffold was identified by screening a small library of non-peptidic compounds. This initial hit was optimized by solution-phase synthesis and screening of a focused library of compounds bearing this scaffold. This led to the identification of a novel, uncompetitive inhibitor (1a40, IC50=5.41±0.45 μM) of WNV NS2B–NS3 protease. Molecular docking of this chiral compound onto the WNV protease indicates that the S enantiomer of 1a40 appears to interfere with the productive interactions between the NS2B cofactor and the NS3 protease domain; (S)-1a40 is a preferred isomer for inhibition of WNV NS3 protease.