Dengue virus (DENV) infection is a worldwide public health concern infecting approximately 400 million individuals and about 40,000 mortalities yearly. Despite this, no licensed or readily available antiviral medication is currently available specifically for DENV infection, and therapy is typically symptomatic. Therefore, the objective of the study is to investigate the antiviral activity of Beta vulgaris L. phytoconstituents against DENV-2 targeting NS3 protein. The antiviral activity of phytochemicals was examined through virtual ligand-based screening, antiviral inhibition assays, dosage response assays, western blotting analysis and MD simulations. We conducted toxicological, and pharmacokinetic analysis to assess plant-based natural compounds' efficacy, safety, and non-toxic doses. Molecular docking and MD simulation results revealed that the nonstructural protein NS3 is the primary target for Betanin and Glycine Betaine against the Dengue virus. Betanin and Glycine betaine were initially studied for their non-toxic doses in HeLa, CHO, and Vero cells via MTT assay. HeLa cells were transiently transfected with cloned vector pcDNA3.1/Zeo(+)/DENV-2 NS3 along with non-toxic doses (80 μM- 10 μM) of selected phytochemicals. The dose-response assay illustrated the downregulated expression of the DENV-2 NS3 gene after the post-treatment of Betanin (IC50 =4.35 μM) and Glycine Betaine (IC50 = 4.49 μM). Betanin (80 μM- 10 μM) was found to inhibit the NS3 protein. These results suggested the down regulated expression of DENV-2 NS3 protein at mRNA level as well as protein level depicting the DENV replication inhibition. Based on our study findings, NS3 protease is depicted as distinctive DENV-2 inhibitor target. We will channel our study further into in vitro characterization employing the mechanistic study to understand the role of host factors in anti-flavi therapeutic.