Natural killer (NK) cells are critical components of the innate immune system, capable of rapidly identifying and eliminating virus-infected and tumor cells without prior antigen sensitization. This rapid cytotoxic response is regulated by a balance between activating and inhibitory receptor signals, a mechanism known as “missing self.” Inhibitory receptors recognize HLA class I molecules, preventing unwanted activation against healthy cells, while activating receptors detect stress-induced ligands on abnormal cells, triggering the release of perforin and granzyme B. Recent evidence shows that inhibitory receptor-HLA class I interactions are peptide-dependent, highlighting a complexity similar to T cell receptor recognition. Alterations in the antigen processing and presentation pathway in cancer cells can modify the immunopeptidome, enhancing NK-mediated cytotoxicity. To assess NK cell function and receptor-ligand interactions, degranulation and killing assays are widely employed. These methods are applicable to both primary NK cells and established NK cell lines, which differ in receptor expression and cytotoxic capabilities. Strategic selection of NK models and target systems allows for in-depth studies of NK cell biology and supports the development of NK cell-based immunotherapies.