Dengue virus (DENV) remains a major global health concern, with no approved antiviral treatments or effective vaccines available to date. The non-structural 5 (NS5) protein, which is the largest and most conserved non-structural protein encoded by orthoflaviviruses, plays a critical role in viral replication, making it an attractive target for drug development. This dissertation provides structural and dynamic insights into the dengue virus NS5 protein to guide structure-based drug design strategies. Using a combination of biophysical, structural and computational approaches, this dissertation research elucidates dengue NS5’s interactions with the viral promoter stem-loop A (SLA) at the 5’-untranslated region of the viral genome. Findings from this dissertation research reveal that SLA binding induces significant conformational rearrangements in NS5, particularly interdomain coordination between its methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domain. CryoEM single particle analysis data further confirm SLA’s conserved binding mode across different DENV serotypes, highlighting a key mechanism of viral replication. Additionally, this dissertation employs a Site Identification by Ligand Competitive Saturation (SILCS) computer-aided drug design (CADD) approach to identify novel allosteric binding sites on DENV NS5, facilitating the discovery of potential small molecule inhibitors. A screening cascade combining in silico and in vitro biophysical techniques has led to the identification of promising candidate compounds with antiviral activity against NS5. Future directions include expanding the structural characterization of NS5 across the four DENV serotypes, developing functional assays to assess NS5 enzymatic activity, and improving computational drug screening methods using machine-learning based algorithms for hotspot ranking. By advancing our understanding of DENV NS5’s conformational landscape and its interactions with viral RNA elements, this dissertation provides a foundation for targeted antiviral strategies, strengthening drug discovery efforts against the dengue virus.