Abstract: In this study, a detailed experimental characterization of the relative ion abundance bias and the ion fragmentation in an ion funnel trap were reported by using both cytochrome C and ubiquitin samples in a tandem ion funnel hybrid ion mobility spectrometer/quadrupole time-of-flight mass spectrometer (IMS/QTOF MS). Specifically, signals of multiple interfering ions appeared in the MS spectra acquired at a relatively short ion filling time of the ion funnel trap for both selected protein samples, while they were absent at longer ion filling time. Further MS/MS studies confirmed that these interfering ions are derived from the fragmentation of the protein ions occurring before the IMS drift tube. The possible reasons for the ion fragmentation, including the ion fragmentation at the exit of the inlet capillary and the radio frequency (RF) heating in two ion funnels, were systematically studied. By carefully adjusting the voltages on each relevant component, the experimental results demonstrated that the ion fragmentation indeed occurs in the ion funnel trap due to excessive RF heating when the RF voltage was higher than 130 V. Therefore, it is extremely important to set the reasonable operation conditions for the ion funnel trap to avoid both ion fragmentation and ion storage bias in IMS/QTOF MS.