Gut microbial metabolites generated through interactions with dietary constituents are important modulators of host metabolism and physiology. Our understanding of diet–microbiome–host interactions is hampered by the limited tools to broadly identify microbial species that take up nutrients and their products. BioOrthogonal labeling-Sort-Sequence-Spectrometry (BOSSS) overcomes these challenges by leveraging orally introduced alkyne-modified lipids that permit identification of gut microbes that take up dietary lipids.