Natural Killer T (NKT) cells have gained widespread attention among immunologists because of their distinct ability to regulate anti-tumor responses and to influence the outcome of infections or autoimmunity. Type I (also called invariant) NKT cells (iNKT) are best characterized mainly because of the availability of lipid antigen-loaded CD1d-tetramer detection reagents. Human iNKT cells present important phenotypic differences relative to their murine counterpart, restricting the extrapolation of findings from experimental murine models to human health and disease states. Particularly, the ontogeny and early life phenotype of iNKT cells largely differ between human and mice, indicating divergent functional properties between species. The high therapeutic potential offered by manipulation of iNKT cells in disease warrants a better understanding of human iNKT cell biology. Here, we discuss characteristics of human iNKT cells and present an efficient and rapid method for their ex vivo purification and characterization