Rationale Isomer metabolites are involved in metabolic pathways, and their characterization is essential but remains challenging even using high-performance analytical platforms. The addition of ion mobility prior to mass analysis can help to separate isomers. Here, the ability of a recently developed trapped ion mobility spectrometry system to separate metabolite isomers was examined. Methods Three pairs of estrogen isomers were studied as a model of isomeric metabolites under both negative and positive electrospray ionization (ESI) modes using a commercial trapped ion mobility spectrometry-TOF mass spectrometer. The standard metabolites were also spiked into human urine to evaluate the efficiency of trapped ion mobility spectrometry to separate isomers in complex mixtures. Results The estradiol glucuronide isomers (E2β-3G and E2β-17G) could be distinguished as deprotonated species, while the estradiol epimers (E2β and E2α) and the methoxyestradiol isomers (2-MeO-E2β and 4-MeO-E2β) were separated as lithiated adducts in positive ionization mode. When performing analyses in the urine matrix, no alteration in the ion mobility resolving power was observed and the measured collision cross section (CCS) values varied by less than 1.0%. Conclusions The trapped ion mobility spectrometry-TOF mass spectrometer enabled the separation of the metabolite isomers with very small differences in CCS values (ΔCCS% = 2%). It is shown to be an effective tool for the rapid characterization of isomers in complex matrices.