The viral NS2B-NS3 protease is a promising drug target to combat dengue virus (DENV) and other emerging flaviviruses. The discovery of novel DENV protease inhibitors with antiviral efficacy is hampered by the low predictive power of biochemical assays. We herein present a comparative evaluation of biochemical DENV protease assay conditions and their benchmarking against antiviral efficacy and a protease-specific reporter gene assay. Variations were performed with respect to pH, type …