Dengue virus (DENV) is an insect borne virus classified in the family Flaviviridae. DENV is the most widespread arthropod borne virus across the globe and according to latest epidemiological reports dengue fever (DF) is one of the major cause of concern for global health.Amongst all four DENV serotypes predominately circulating serotypes in Pakistan are DENV2 and DENV3. RNA interference (siRNA and miRNA) is at the forefront of molecular approaches for inhibitory studies. The present study was aimed at inhibition of both these serotypes via siRNA interference; moreover structural genes were cloned for insilico analysisto facilitate strategies for the development of vaccines against DENV. Both DENV2 and DENV3 were targeted by synthetic siRNAsdesigned against conserve regions in the genome. Six siRNAs were designed against both DENV2 and DENV3 from three vital regions of genome 5''untranslated region (5''UTR), 3''UTR and structural genes. Transfections were performed by Lipofectamine and level of inhibition was observed by performing several viral titer estimation assays. Results showed thatDENV3UTR3''siRNA2 targeting 3'' UTR was able to inhibit DENV3 replication.The replication cycle of DENV has revealed that in human Dendritic cells (DCs),Furin enzyme converts the immature DENV pre-membrane protein (prM) to mature M protein assisted by change in cellular pH.Therefore, in the later part of study fully mature DENV2 were allowed to infect and later targeted by RNAi. Results from this study showed that siRNA (DENV2SsiRNA2) targeting the M region of genome was able to drastically knock down DENV2 in Vero-81 cells. Substantial inhibition was also seen by siRNA (DENV2UTR3''siRNA2) targeting 3'' UTR region ofDENV2 genome. Structural genes were cloned from locally isolated DENV2 serotype. All three genes (Capsid, prM and Envelope) were subjected to insilico analysis involving construction of phylogenetic trees and prediction of epitopic domains. It was observed that both Pakistani and Indian DENV2 share a strong sequence similarity. Inhibition of both DENV serotypes via RNAi highlights the effectiveness of molecular approaches for anti-dengue drug development. Moreover, findings of insilico study of Pakistani DENV2 serotype will assist in the development of regionally targeted vaccine for densely populated South Asian region.