In this paper, high-resolution nano-electrospray ionization-trapped ion mobility spectrometry coupled to mass spectrometry (nESI-TIMS-MS) is used for the study of hydroxylated polybrominated diphenyl ether (OH-PBDE) metabolites. In particular, experimental ion-neutral collision cross sections (CCS) were measured for five structural OH-PBDE isomers using TIMS-MS. Candidate structures were proposed for each IMS band observed in good agreement with the experimental CCS measurements (5% error). The analytical power of TIMS-MS to baseline and partially separate structural isomers of OH-BDE in binary and ternary mixtures is shown for single charge species with a mobility resolving power of RIMS ~ 400. This work provides the proof of concept for the analysis of low concentration OH-PBDE in environmental samples based on accurate collision cross section and mass measurements without the need for derivatization and pre-fractionation protocols, thus significantly reducing the cost and analysis time.