Transient receptor potential melastatin 2 (TRPM2) is a multifunctional non-selective Ca2+-permeable cation channel expressed in numerous immune cells. 2′-Deoxy-adenosine diphosphoribose (2d-ADPR) has been identified as a superagonist of TRPM2 channels that induces higher whole-cell currents and requires a lower intracellular Ca2+ concentration for activation. 2d-ADPR can be produced from 2′-deoxy-nicotinamide adenine dinucleotide (2d-NAD) by the enzyme CD38 in vitro under physiological conditions. Here, we describe a two-dimensional HPLC method, suitable for the quantification of endogenous 2d-ADPR and 2d-NAD. The results demonstrated that 25 million Jurkat T cells per sample were sufficient to quantify endogenous 2d-ADPR and 2d-NAD.