Natural Killer T (NKT) cells are a subset of T lymphocytes that recognize a wide variety of lipid antigens presented by CD1 molecules. NKT cells exhibit rapid activation after recognition of cognate antigens, secrete abundant amounts of T helper (Th) 1, Th2, and Th17 cytokines within hours of activation and shape the immune response through subsequent activation of dendritic, NK, T and B cells. NKT cells therefore play central roles in antimicrobial and anticancer immunity and in modulation of various autoimmune disorders. Consequently, recent research has focused on the discovery of microbial and self-antigens involved in NKT cell activation. In this chapter, we discuss different strategies for studying antigen recognition by NKT cells including CD1d tetramer-based approaches and in vitro assays characterizing NKT cell activation in response to lipid antigen presentation. While toll-like receptor (TLR) agonists and cytokines such as IL-12 are critical for NKT cell activation in vivo, particularly in the context of microbial infection, methods for detection of TLR- and cytokine-dependent NKT cell activation will not be discussed in this section.