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Efforts to enhance wellness and ameliorate disease via nutritional, chronobiological, and pharmacological interventions have markedly intensified interest in ketone body metabolism. The two ketone body redox partners, acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB) serve distinct metabolic and signaling roles in biological systems. A highly efficient, specific, and reliable approach to simultaneously quantify AcAc and D-βOHB in biological specimens is lacking, due to challenges of separating the structural isomers and enantiomers of βOHB, and to the chemical instability of AcAc. Here we present a single UPLC-MS/MS method that simultaneously quantifies both AcAc and βOHB using independent stable isotope internal standards for both ketones. This method incorporates one sample preparation step requiring only 7 min of analysis per sample. The output is linear over three orders of magnitude, shows …