The immune system serves as a barrier limiting the initiation and progression of new tumors. Among its cellular components, CD8+ T cytotoxic cells are integral to the immune response against cancer. These cells can identify and combat tumor cells by releasing lytic granules, thereby driving the adaptive immune response. However, their function can be hindered by several factors such as immunosuppression in the tumor microenvironment, T cell exhaustion, and immunosenescence. Consequently, dissecting these mechanisms is essential to fully comprehend the immune response against cancer cells and can aid in tailoring future antitumor immunotherapies. In this chapter, we outline a straightforward method for isolating CD8+ T cells from the mouse spleen. These cells can then be further analyzed or utilized in in vitro functional studies, such as coculture with target tumor cells and assessing their cytotoxic activity and cytokine production profiling.