Human natural killer (NK) cells, a subset of peripheral blood lymphocytes that lack a T- or B-cell receptor, play a crucial role in the innate immune response to viruses and malignant cells. NK cells differentiate infected or malignant cells from normal cells by a complex balance between activating and inhibitory receptor-ligand interactions. Unlike T cells, NK cells do not proliferate in vitro in response to simple crosslinking of a single activating receptor. While many methods to study T-cell function and phenotype can also be applied to NK cells, this chapter addresses methods that are unique to the preparation and assessment of human NK cells for immunotherapy.