The human gut microbiome is a complex ecosystem that contributes to the health of the host by transforming nutrients from the diet. Many of these interactions are mediated through the exchange of metabolites between the microbes and the host. Here, we describe two complementary targeted metabolomics techniques, liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR), to monitor specific changes in metabolite abundance in samples from human urine, fecal, and in vitro GI colon model systems. This approach offers the specificity and sensitivity of LC-MS quantification with an understanding of broad changes in metabolite abundance by NMR that together can aid in understanding how the gut microbiome metabolizes different dietary nutrients.