Natural killer (NK) cells are innate lymphocytes that can recognize and rapidly eliminate malignantly transformed or virally infected cells without requiring prior sensitization or clonal expansion. In the setting of adoptive cancer immunotherapy with chimeric antigen receptor (CAR)-engineered lymphocytes, NK cells represent a promising alternative to T cells, since they can be transferred across HLA barriers without the risk of inducing graft-versus-host disease, are not prone to cause cytokine release syndrome or neurotoxicity, and retain endogenous natural cytotoxicity in addition to CAR-mediated target cell killing. Different sources of NK cells are actively pursued for the development of cancer immunotherapies, including continuously expanding human NK cell lines. Thereby, NK-92 cells have not only been instrumental to initially establish the concept of cellular off-the-shelf therapeutics but are also the basis of many ongoing approaches to advance functionally enhanced derivatives to a clinical setting. Here, we provide an overview on human NK cell lines and summarize their preclinical and clinical development for cancer immunotherapy, with a particular focus on NK-92 as the most widely used and best studied NK cell line to date.