Cancer immunotherapy approaches aim to instruct the immune system to eliminate tumor cells. Immune checkpoint blockade (ICB) therapies, such as anti-CTLA-4 and anti-PD-1, have significantly advanced the field, and other immunotherapeutic modalities are constantly being developed and tested in pre-clinical and clinical studies. One of the most important outcomes of cancer immunotherapy is efficient elimination of tumor cells by cytotoxic CD8+ T cells. Thus, accurate measurement of tumor cell sensitivity to T-cell-mediated killing is crucial for improving treatment development. This paper presents a flow cytometry-based protocol to assess antigen-specific CD8+ T-cell cytotoxicity using ovalbumin (OVA)-specific CD8+ T cells from OT-1 TCR transgenic mice and OVA-presenting tumor cell lines (either transduced with OVA or pulsed with a class-I-MHC-restricted OVA-derived peptide). This method minimizes contribution from non-antigen-mediated killing into readouts by incorporating non-target cells (not expressing/presenting-OVA cells) into co-cultures. The procedure also allows for the evaluation of T-cell-mediated cytotoxicity under various conditions and enables concurrent immunophenotyping of effector T cells and target tumor cells if desired. The protocol can be easily customized, offers advantages over methods that rely on luciferase-expressing tumor cell lines and provides versatility in detecting multiple parameters, making it a useful and valuable tool for advancing cancer immunotherapy research.