Abstract: A gas phase ion separation field asymmetric waveform ion mobility spectrometry-ion mobility spectrometry-quadrupole time-of-flight mass spectrometry (FAIMS-IMS-QTOF MS) system that integrates a high-sensitivity and high-resolution racetrack field asymmetric waveform ion mobility spectrometry (r-FAIMS) with an ion mobility spectrometry quadrupole time-of-flight mass spectrometry (Agilent 6560 IMS-QTOF MS) was applied to probe the conformational diversity of myoglobin ions. A charge state envelope of +12 to +25 for the myoglobin ions was observed in the acquired MS spectrum. For myoglobin ions with charge states ranging from +12 to +14, multiple IMS peaks were clearly observed in the acquired IMS spectra, whereas only one peak was observed in the corresponding FAIMS spectra. In addition, multiple FAIMS peaks can be easily observed for myoglobin ions with charge states from +15 to +25 in the corresponding FAIMS spectra, while only one peak was observed in the corresponding IMS spectra. Gas phase ion separation in FAIMS and IMS were thus highly orthogonal. However, the ions from different FAIMS peaks were shown to have very close drift times in the acquired IMS spectra, which implied that these ions from different FAIMS peaks may have essentially the same collisional cross-section areas. To verify that these FAIMS peaks, corresponding to different compensation voltages, for myoglobin ions with charge states of +15 to +25 indeed represent different conformers, the compensation voltage (CV)-selected myoglobin ions were further subjected to ion fragmentation (MS/MS) analysis using the QTOF MS. The MS/MS analysis clearly demonstrated that the product ion spectra for myoglobin ions of the same charge state from different FAIMS CV peaks are highly distinct under the same MS/MS operating conditions (i.e. under the same collision energy). The results from MS/MS analysis thus convincingly proven that the protein ions from different FAIMS peaks are different conformers. Unlike other analytical platforms, the FAIMS-IMS-QTOF MS system used in this study allows the simultaneous highly efficient FAIMS and IMS separation of protein structural isomers, a detailed IMS measurement of collisional cross-section area for each isomeric protein and a direct confirmation of isomeric proteins via MS/MS. Therefore, this system can be a very powerful new tool for the structural characterization of isomeric protein ions in general.