CRISPR-Cas9, an RNA-guided genome editing technology, is revolutionizing cell biology due to the ease and efficiency by which it enables genetic manipulation of mammalian cells. Through targeted modification of specific genes or regulatory regions, researchers can now rapidly generate precise genetic models to study normal and diseased cell physiology. Beyond genetic manipulation for research purposes, CRISPR-Cas9 genome editing also holds great potential for therapeutic applications, including immunotherapy and regenerative medicine. This protocol provides instructions for CRISPR-Cas9 genome editing of human primary T cells, including the isolation and activation of primary human T cells, preparation of a CRISPR-Cas9 RNP complex, delivery of an RNP complex into activated primary T cells using electroporation, and methods to evaluate genome editing efficiency.For a discussion of alternative strategies and expected outcomes, and protocol modifications, please see: Technical Bulletin: Genome Editing of Human Primary T Cells.