Natural killer T (NKT) cells are a distinct T-cell sublineage, originally named because of their coexpression of an alphabeta T cell antigen receptor (TCR) characteristic of T lymphocytes, and NK1.1, a C-type lectin expressed by natural killer (NK) cells. NKT cells use their TCR to recognize glycolipids bound to or presented by CD1d. Until recently, most studies used the synthetic glycolipid alpha-galactosylceramide (alphaGalCer) to activate these lymphocytes, and very little was known about the natural antigens recognized by NKT cells. Given the pivotal role played by the NKT cells in many immune responses, including antimicrobial responses, tumor rejection, and the development of autoimmune diseases, the identification of the natural antigens recognized by these cells, and analogs that may alter their cytokine production, are goals of primary importance. This chapter discusses methods that can be used to assess the potency of potential glycolipid antigens for this unique population of T lymphocytes, including methods for in vitro NKT cell activation and expansion, in vivo activation, and measurement of their avidity for different antigens.