Dengue is a severe mosquito-borne viral infection causing half a million deaths annually.Dengue virus NS2B/NS3 protease is a validated target for anti-dengue drug design. A series of hitherto unreported 3,5-bis(arylidene)-4-piperidones analogues 4a–4j were synthesized and screened in silico against DENV2 NS2B/NS3 protease to elucidate their binding mechanism and orientation around the active sites. Results were validated through an in vitro DENV2 NS2B/NS3 protease assay using a fluorogenic Boc-Gly-Arg-Arg-AMC substrate. Nitro derivatives of 3,5-bis(arylidene)-4-piperidones(4e and 4j) emerged as promising lead molecules for novel protease inhibitors with an IC50 of 15.22 and16.23 mmol/L, respectively, compared to the standard, panduratin A, having IC50 of 57.28 mmol/L. Dengue is a severe mosquito-borne viral infection causing half a million deaths annually. Dengue virus NS2B / NS3 protease is a validated target for anti-dengue drug design. A series of hitherto unreported 3,5-bis (arylidene) -4-piperidones analogues 4a-4j were synthesized and screened in silico against DENV2 NS2B / NS3 protease to elucidate their binding mechanism and orientation around the active sites. Results were validated through an in vitro DENV2 NS2B / NS3 protease assay using a fluorogenic Boc-Gly-Arg- Arg-AMC substrate. Nitro derivatives of 3,5-bis (arylidene) -4-piperidones (4e and 4j) emerged as promising lead molecules for novel protease inhibitors with an IC50 of 15.22 and 16.23 mmol / L, respectively, compared to the standard, panduratin A, having an IC50 of 57.28 mmol / L.