RNA interference (RNAi) is a highly conserved, specific and selective biological response of gene regulation mechanism and has been widely exploited for designing novel drug targets to treat infections caused by pathogens including viruses. In the previous study, one of the promising sh-5b RNA (short hairpin RNA, sh- 5b) construct was targeted to the conserved sequence within the 5¹-NTRs (non-translated regions) of all Dengue serotypes. The current study report the stability (up to 24 months) of recombinant adenovirus (rAd) engineered to express sh-5b RNA for DENV-2 replication inhibition. Stability study was done by gene specific PCR, where as the effect of sh-5b RNA in attenuating DENV-2 replication was further validated by One step RT-PCR by TITAN. The rAds harboring sh-5b RNA to target dengue serotypes might serve as an efficient alternative to conventional drugs for therapeutic intervention to treat DENV infections in humans.