Infection rate from the viral pathogen West Nile Virus (WNV) is a true health concern, wherein some cases outbreaks are observed around the globe. Because the virus can be neuroinvasive if left untreated, it can lead to cases of meningitis and/or encephalitis. WNV belongs to the Flaviviridae, a family of viruses containing an error-prone RNA polymerase giving rise to frequent mutations resulting in immune escape and resistance. Currently no vaccines or antiviral treatments are available for WNV, highlighting the need for further investigation to discover new antivirals, and the tools necessary to find them. One of the central steps in the life cycle of WNV is the processing of its proteome, carried out by host and viral proteases. We focus on the viral protease (PR) encoded by the viral genome, since it is the first cleavage event necessary for viral infection to ensue. For this purpose a cell-based assay was developed to monitor the activity of the viral protease. The strategy utilized exploits the auto-catalytic property of the viral NS3 PR in a fusion context with the well-studied yeast transcription factor Gal4. The DNA-binding domain (DBD) and transcriptional-activation domain (TAD) of Gal4 are separated by NS3 and its cofactor NS2B. Upon translation of this fusion in the cell, monitoring of cleavage events is evaluated based on Gal4 promoter-driven expression of the reporter Green Fluorescent Protein (GFP), which can only be achieved in the presence of an intact DBD/TAD fusion. As a result, two distinct conditions can arise: (1) In the presence of cleavage, the Gal4/PR fusion is destroyed due to the auto-catalytic activity of wild-type PR, resulting in no GFP induction. (2) In the absence of cleavage due to a mutated or inhibited PR, the DBD/TAD fusion remains intact, traveling to the nucleus and inducing GFP expression. The assay will be further developed to robustly monitor PR activity through retroviral technology in an inducible manner to protect the cells from the possible cytotoxic effects of PR overexpression. Utilizing flow cytometry, the cell-based assay provides a platform for the high-throughput screening for novel inhibitors of WNV.