Hepatic mitochondria play a central role in the regulation of intermediary metabolism and maintenance of normoglycemia, and there is great interest in assessing rates of hepatic mitochondrial citrate synthase flux (V_CS) and pyruvate carboxylase flux (V_PC) in vivo. Here, we show that a positional isotopomer NMR tracer analysis (PINTA) method can be used to non-invasively assess rates of V_CS and V_PC fluxes using a combined NMR/gas chromatography-mass spectrometry analysis of plasma following infusion of [3-¹³C]lactate and glucose tracer. PINTA measures V_CS and V_PC fluxes over a wide range of physiological conditions with minimal pyruvate cycling and detects increased hepatic V_CS following treatment with a liver-targeted mitochondrial uncoupler. Finally, validation studies in humans demonstrate that the V_PC/V_CS ratio measured by PINTA is similar to that determined by in vivo NMR spectroscopy. This method will provide investigators with a relatively simple tool to non-invasively examine the role of altered hepatic mitochondrial metabolism.