Natural killer (NK) cells have emerged as promising candidates for novel immunotherapy strategies against various malignancies. Their unique ability to recognize and eliminate tumour cells without prior sensitization, coupled with the secretion of pro-inflammatory cytokines such as interferon-gamma and tumour necrosis factor, position them as promising agents in cancer therapy. Adoptive NK cell transfer has shown particular promise in haematological malignancies, where NK cell infusions could achieve remission in a high proportion of patients. Moreover, the possibility to engineer NK cells to express chimeric antigen receptors can further enhance their efficacy, thereby broadening their applicability to include solid tumours. Ongoing research is crucial to optimize NK cell therapies and enhance their efficacy to expand their clinical applications. However, this research hinges on robust protocols and experimental methodology for the isolation, expansion, and genetic engineering of NK cells. In an attempt to set up a standardized protocol for NK cell isolation and expansion, we present a thoroughly tested and validated protocol that can produce highly pure, viable, and potent NK cells that can be used for research and development of NK cell therapies. The protocol is highly reproducible, closely aligned to comply with Good Manufacturing Practice regulations, and tested for scalability to produce NK cells at clinically relevant dosages to support the development of off-the-shelf NK products.