T cells are a type of white blood cell that can mount memory responses against pathogens, tumors, and other threats. They are an essential component of the immune system, conferring immune memory against harmful entities and promoting tolerance to self-antigens. Immunotherapies for cancer leverage T cell activation for tumor clearance, highlighting their importance. Vaccinations prime and enhance T cell memory responses against infectious diseases, while regulatory T cells help prevent autoimmune diseases. A thorough comprehension of T cell biology holds the key to future therapies for these and various other conditions. This Methods Collection aims to assist the research community by providing clear and concrete visual protocols documenting current methods used to assess T cell migration, function, memory, and other facets of T cell biology. Providing a compendium of methods for investigating T cell biology in the context of different homeostatic and disease states is crucial for upholding best practices in the field.