Technical Abstract: An ultra-high performance liquid chromatography-ion mobility- quadrupole time-of-flight mass spectrometry (UHPLC-IM-QTOF-MS) method was developed for profiling the indole alkaloids in yohimbe bark. Many indole alkaloids with the yohimbine core structure, plus methylated, oxidized, and reduced species, were characterized. Common fragments and mass differences are described. It was shown that the use of IM could provide another molecular descriptor; i.e., molecular shape by rotationally averaged collision cross-section was of great value for identification of constituents where reference materials are usually not available. Using the combination of high resolution (~40000) accurate mass measurement with time aligned parallel (TAP) fragmentation, MSE (where E represents collision energy), ion mobility mass spectrometry (IMS), and UPLC chromatography, a total of 56 indole alkaloids were characterized and a few new yohimbine-like alkaloids are reported for the first time.