In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λ max for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.Key words dengue virus type 2; envelope protein; tryptophan assay; fluorescence spectroscopy More than 2.5 million people are at risk of dengue infections in over 100 countries, with several hundred thousand cases of life threatening dengue hemorrhagic fever (DHF)/ dengue shock syndrome (DSS) occurring annually.1) Currently, there is no commercially available vaccine or antiviral agent against dengue virus (DENV) infections. In the efforts towards developing an anti-viral treatment for dengue, a simple tryptophan fluorescence quenching screening assay aimed at identifying dengue EIII protein inhibitors was developed.DENV is a positive-sense, single stranded RNA virus belonging to the Flaviviridae family. The DENV consists of four antigenically distinct serotypes (serotypes 1-4). The glycosylated envelope (E) protein is a type 1 integral membrane protein, and makes up the major structural protein present on the surface of the mature dengue virions. It plays an important role in viral infection via virus attachment to cell-surface receptors and mediates viral-cell membrane fusion. The E protein is the major target for protective antibodies against dengue.2) It has been demonstrated that the mature DENV E protein in the pre-fusion state forms homodimers in an antiparallel manner (head to tail orientation), which transforms to its trimeric form in the post-fusion state.2) The stability and function of the DENV E protein during conformational transition from dimeric to trimeric form is controlled by the change in pH. Each monomer is folded in three distinct domains, with the central domain (domain I) bordered by the immunoglobulin-like C-terminal domain (domain III) and the dimer...