Flaviviral NS2B/NS3 heterocomplex serine proteases are a primary target for anti-flavivirus drug discovery. To gain insights into the enzymatic properties and molecular determinants of flaviviral NS2B/NS3 protease substrate specificity in host cells, we developed and applied a novel series of membrane-anchored red-shifted fluorescent protein substrates to detect West Nile virus (WNV) NS2B/NS3 endoproteolytic activity in human cells. The substrate consists of a fluorescent reporter group (DsRed) tethered to the endoplasmic reticulum membrane by a membrane-anchoring domain. Between the two domains is a specific peptide linker that corresponds to the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 protein junctions within the WNV polyprotein precursor. When the protease cleaves the peptide linker, the DsRed reporter group is released, changing its localization in the cell from membrane-bound punctate perinuclear to diffuse cytoplasmic. This change in protein location can be monitored by fluorescent microscopy, and cleavage products can be quantified by Western blotting. Our data demonstrate the robustness of our trans-cleavage fluorescence assay to capture single-cell imaging of membrane-associated WNV NS2B/NS3 endoproteolytic activity and to perform in-cell selectivity profiling of the NS2B/NS3 protease. Our study is the first to provide cellular insights into the biological and enzymatic properties of a prime target for inhibitors of WNV replication.