Natural killer (NK) cells are major actors of innate immune responses against viruses, bacteria, parasites, and other mediators of pathology such as malignant transformation. These cells are also directly implicated in the link between innate and adaptive immunity, shaping T-cell responses. It is now obvious that manipulation of this lymphocyte subset could be the basis of new therapeutic approaches for cancer and/or pathogen-driven pathology. Hence, techniques enabling the phenotypic and functional analysis of patient NK cells are of major importance. In this chapter, we present an extensive immunophenotyping of patient NK cells as well as a recently described method to assess NK cell functional activity at the single-cell level.