Hydrodynamic tail vein (HTV) delivery is a simple and rapid tail vein injection method of a high volume of naked plasmid DNA resulting in high levels of foreign gene expression in organs, especially the liver. Compared to other organs, HTV delivery results in more than a 1000-fold higher transgene expression in liver. After being bitten by malaria-infected mosquitoes, malaria parasites transiently infect the host liver and form the liver stages. The liver stages are known to be the key target for CD8+ T cells that mediate protective anti-malaria immunity in an animal model. Therefore, in this study, we utilized the HTV delivery technique as a tool to determine the in vivo cytotoxic effect of malaria antigen-specific CD8+ T cells. Two weeks after mice were immunized with recombinant adenoviruses expressing malarial antigens, the immunized mice as well as naïve mice were challenged by HTV delivery of naked plasmid DNA co-encoding respective antigen together with luciferase using dual promoters. Three days after the HTV challenge, non-invasive whole-body bioluminescent imaging was performed. The images demonstrate in vivo activity of CD8+ T cells against malaria antigen-expressing cells in liver.