Cytotoxic CD8⁺T lymphocytes (CTLs) represent a crucial component of the adaptive immune system and play a prominent role in the anti-tumor immune responses of both mice and humans. Cytotoxic CD8⁺T cells are responsible for the lysis of cells expressing peptides associated with MHC class I molecules and derived from infection with a pathogen or from mutated antigens. In order to quantifyin vivothis antigen-specific CD8⁺T cell killing activity, we use thein vivokilling assay (IVKA). Here, we describe the protocol for the lysis of cells expressing a CD8⁺T cell melanoma epitope of the hgp100_25-33protein (KVPRNQDWL). C57BL/6 recipient mice, receive first target cells, prepared from naive congenic (CD45.1) C57BL/6 spleen cells pulsed with the hgp100_25-33peptide and labeled with CFSE and of non-pulsed control cells labeled with Brilliant violet. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSE cells and Brillant Violet (BV) cells. The percentage of lysis is calculated by the difference between CFSE versus BV. Measuring the ability of antigen-specific CD8⁺T cells to lyse their antigenin vivois very important to evaluate the adaptive cytotoxic response induced against a pathogen or a tumor antigen.