Diacylglycerols (DAGs) are important lipid intermediates in cellular trafficking and signaling. Their concentrations are altered in diabetes, cancer, and other disease states. Quantification of DAGs in biological samples may provide critical information to uncover molecular mechanisms leading to various cellular functional disorders. Recent advances in lipidomics using mass spectrometry have greatly accelerated global lipid analysis and quantification. Quantification of DAGs by electrospray mass spectrometry (ESI/MS), however, is challenged by the absence of a permanent charge on the molecule, its low proton affinity and acidity, and its low abundance under normal biological conditions. We describe here the introduction of a quaternary ammonium cation to DAG molecules, using N-chlorobetainyl chloride, to afford a derivatized DAG that gives 2 orders of magnitude higher signal intensities than their underivatized sodium adducts. A linear calibration curve in which peak intensity ratios are plotted versus molar ratios can be achieved by using ESI/MS with dilauroyl glycerol as the internal standard. Employing this new approach to this analyte, we found a 9-fold increase of total DAGs in the livers of obese db/db mice as compared to their heterozygous lean controls. This proven strategy can be used to detect and quantify DAG molecular species from biological samples using ESI/MS after one-step derivatization.