The major challenge in CD1 antigen discovery is designing a system to unequivocally identify rare antigenic lipids from among abundant and structurally similar endogenous or exogenous lipid ligands that also bind CD1 proteins. We invented the T cell receptor (TCR) Trap, which takes advantage of specific TCR binding to the CD1-antigen complexes to pull out and then sensitively characterize antigenic lipids by mass spectrometry methods. Applying this method to CD1a autoreactive T cells, we discovered a highly unexpected antigen recognition pattern whereby many structurally unrelated lipids could mediate CD1a and TCR binding, leading to the absence of interference model. For CD1d-iNKT cells, the TCR trap identified a single type of α-linked monohexosylceramide in the TCR-lipid-CD1d ternary complex, bypassing the usual two-stage discovery method in which compounds are first tested for T cell activation and then later for biochemical content. Here we characterize CD1b ligands, which are all molecules eluted from cellular CD1b as well as the subset of ligands that allow ternary complexes of CD1b-lipid-TCRs to form. First, we developed a quantitative mass spectrometry method to determine the number and absolute quantities of lipid ligands bound to CD1b. Based on nominal mass and collisional pattern, we next deduced the molecular structure and exact masses of the lipid ions, emphasizing the role of phosphatidylglycerol and other phospholipids.  Third, we confirmed the identity of each lipid using a quadrupole time-of-flight mass spectrometer equipped with high performance liquid chromatography, which provides a retention time and the accurate mass. Our data demonstrate a clear correlation between endogenous lipids eluted from CD1b and the cellular lipids of the expression system. Our method provides for the first time, a quantitative approach to determine the absolute amount of ligands bound to CD1b and  antigens trapped in TCR and CD1 complexes.