The homeostasis of bile acid (BA)-submetabolome that is composed by correlating hundreds of BA species contributes a lot to maintaining physiological status. However, it is challenging to understand the transformational rules amongst endogenous BAs, but it is viable to profile the in vitro metabolism of BA analogues, as a compromise approach to isotopic labeling of BAs, to deduce the metabolism of BAs. An attempt is made here to characterize the metabolites of 23-nordeoxycholic acid (norDCA), a deoxycholic acid analogue with a C23-CH2 defect, after in vitro incubation with enzyme-enriched liver subcellular fractions of mouse, rat or human. A predictive multiple-reaction monitoring mode was deployed for sensitive metabolite detection, leading to the capture of twelve metabolites (M1-M12). After putative structural annotation by analyzing MS/MS spectra, special attention was paid to isomeric identification. Dozens of authentic BAs were collected and measured for modeling of the quantitative structure-retention time relationships. Because modifications in LC-MS/MS behaviors in response to C23-CH2 difference were characterized by comparing several pairs, the rules of 14.02 Da shift and 2.4-4.2 min distance were applied to improve identification confidence by matching with several authentic BAs bearing C23-CH2 additions compared to the metabolites. Consequently, confirmative 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 about the correlations between different endogenous BAs and the structural identification strategy offers a promising idea when facing an isomeric discrimination challenge.