The homeostasis of bile acid (BA)-submetabolome that is composed by correlating hundreds of BA species, contributes a lot to maintain physiological status. However, it is challenging to understand the transformation rules amongst endogenous BAs, and it is viable to profile in vitro metabolism of BA analogues, as a compromise approach for isotopic labeling BAs, to deduce that of BAs. An attempt was devoted here to characterize metabolites of 23-nordeoxycholic acid (norDCA), a deoxycholic acid analogue with C23-CH2 defect, after in vitro incubation with enzyme-enriched liver subcellular fractions of mouse, rat or human. Predictive multiple-reaction monitoring mode was deployed for sensitive metabolite detection, leading to capturing twelve metabolites (M1−M12). After putatively structural annotation through analyzing MS/MS spectra, special concerns were paid onto isomeric identification. Dozens of authentic BAs were collected and measured for quantitative structure-retention time relationship modeling. Because LC−MS/MS behavior modifications in response to C23-CH2 difference was characterized by comparing several pairs, the rules of 14.02 Da shift and 2.4−4.2 min distance were applied to advance identification confidence by matching with several authentic BAs bearing C23-CH2 additions compared to the metabolites. Consequently, confirmatively structural identification was achieved for all metabolites. Metabolic pathways in response to M1−M12 were proposed, and hydroxylation, oxidation, epimerization, sulfation, and glucuronidation served as the primary metabolism channels for norDCA. Together, the findings provide meaningful information for the correlations amongst different endogenous BAs and the structural identification strategy offers a promising idea when facing isomeric discrimination challenge.