In the absence of a better alternate, (51)Cr release assay, with its several disadvantages is still the most common method for detection of MHC class I restricted T-cell mediated cytotoxicity. We describe a system in which the T-cell mediated cytotoxicity can be assessed using host-derived cells transfected with a bicistronic vector expressing the specific antigen and a quantifiable reporter as target cells. This overcomes the problems associated with use of radioactivity, pre-loading of target cells with reporter/antigen and the MHC restriction. We used HBV core antigen to prove the concept, as it is an established CTL target. Bicistronic vectors containing HBV core and reporter (EGFP/Fluc) gene were generated and further checked for antigen/reporter expression in human HepG2, mouse fibroblast BALB/c.3T3 and mouse lymphoma A20 cell lines. The effector cells to study the cytolytic activity were generated in vivo using BALB/c mice immunized with antigen expressing DNA clone or protein. The target cells (BALB/c.3T3 and A20) transiently transfected with bicistronic constructs were incubated with effector cells (splenocytes) from immunized mice at a different effector to target ratio. Following incubation the CTL activity was calculated by measuring the reporter luciferase in the remaining viable target cells that inversely correlates with the cytolysis of susceptible cells. The percent specific lysis measured in our assay was compared with conventional (51)Cr release assay to validate this approach. This novel bicistronic vector based cytotoxicity assay demonstrated an easy to perform, antigen-specific and non-radioactive method of determining T-cell mediated cytotoxicity.