Although structural isomers may yield indistinguishable ion mobility (IM) arrival times and similar fragment ions in tandem mass spectrometry (MS), it is demonstrated that post-IM/collision-induced dissociation MS (post-IM/CID MS) combined with chemometrics can enable independent study of the IM-overlapped isomers. The new approach allowed us to investigate the propensity of selected b type fragment ions from AlaAlaAlaHisAlaAlaAla-NH2 (AAA(His)AAA) heptapeptide to form different isomers. Principle component analysis (PCA) of the unresolved post-IM/CID profiles indicated the presence of two different isomer types for b4(+), b5(+), and b6(+) and a single isomer type for b7(+) fragments of AAA(His)AAA. We employed a simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) to calculate the total IM profiles and CID mass spectra of b fragment isomers. The deconvoluted CID mass spectra showed discernible fragmentation patterns for the two isomers of b4(+), b5(+), and b6(+) fragments. Under our experimental conditions, calculated percentages of the "cyclic" isomers (at the 95% confidence level for n = 3) for b4(+), b5(+), and b6(+) were 61 (± 5)%, 36 (± 5)%, and 48 (± 2)%, respectively. Results from the SIMPLISMA deconvolution of b5(+) species resembled the CID MS patterns of fully resolved IM profiles for the two b5(+) isomers. The "cyclic" isomers for each of the two-component b fragment ions were less susceptible to ion fragmentation than their "linear" counterparts.