Bile acids (BAs) as a class of crucial gut microbiota-host cometabolites have received considerable attention for more than a century . As early as the 1960s, gut microbiota was reported to enrich the molecular diversity of BAs in four pathways: deconjugation of glycine or taurine, dehydroxylation, dehydrogenation, and epimerization . Very recently, Quinn et al. presented the fifth gut microbiota-based BA metabolism mechanism by which unique amino acid conjugated cholic acid, due to the discovery of three new amino acid-conjugated bile acids (AA-BAs) synthesized in the gut under the mediation of gut microbiota, including phenylalanocholic acid, tyrosocholic acid, and leucocholic acid . This discovery changed the conventional view that free BAs solely conjugate glycine or taurine in the liver. Our recent study has also discovered and characterized two novel amino acid conjugations (alanine conjugation and proline conjugation) with host BAs in mouse intestinal contents and feces using our developed alternating dual-CE scanning MS approach . These findings shed a light on the exploration of the gut metabolome.