Currently, the assessment of functional immunological relevance is mainly done in animal models. Motivation to work on non-animal methods, or new approach methods (NAM), stems from economical and ethical considerations, and is supported by public pressure. Importantly, the translational gap between results obtained in animal studies and clinical trials in humans (the ‘valley of death’), combined with the reproducibility crisis in science, also provide strong scientific arguments to work on novel, robust, human-based methodology. The field of immunology confronts NAM scientists with specific challenges. Firstly, immunological responses require several cell types in different locations for proper development and take considerable time to develop. Secondly, immunological responses in outbred humans are characterized by genetic and functional variability. Still, the development and application of NAM are increasing rapidly, and the field is moving at such a fast pace that a special issue is timely. Our goal is to provide an overview of the current state-of-the-art regarding new approach methods or non-animal methods (NAM) in immunology. These should be inspired by the desire to mimic in vivo biology and describe e.g. challenges in mimicking immunological structures (like lymph nodes, bone marrow, local immune structures), immunological responses (systemic and local, innate and adaptive, B cells and T cells) and/or immunological processes (like maturation, trafficking, extravasation, immunotoxicity, affinity maturation).