Whereas many prior studies have added exogenous antigens to understand CD1-reactive T cell response, the direct elution of lipids from cellular CD1 proteins provides insight into the types of natural lipids loaded onto CD1 in cells. Using a new lipidomics platform, we have broadly eluted endogenous self ligands from CD1a proteins to determine the dominant structures of lipids bound. Recent work on CD1a has identified patterns of capture based on lipid chain length and saturation, so we used these natural patterns to synthesize optimized ligands for CD1a. Using human CD1a tetramers, we evaluated synthetic compounds for blockade of TCR-CD1a binding to develop new lipids that can be studied for in vivo modulation of T cell responses to CD1a.