The investigation of macromolecular biomolecules with ion mobility mass spectrometry (IM-MS) techniques has provided substantial insights into the field of structural biology over the past two decades. An IM-MS workflow applied to a given target analyte provides mass, charge, and conformation, and all three of these can be used to discern structural information. While mass and charge are determined in mass spectrometry (MS), it is the addition of ion mobility that enables the separation of isomeric and isobaric ions and the direct elucidation of conformation, which has reaped huge benefits for structural biology. In this review, where we focus on the analysis of proteins and their complexes, we outline the typical features of an IM-MS experiment from the preparation of samples, the creation of ions, and their separation in different mobility and mass spectrometers. We describe the interpretation of ion mobility data in terms of protein conformation and how the data can be compared with data from other sources with the use of computational tools. The benefit of coupling mobility analysis to activation via collisions with gas or surfaces or photons photoactivation is detailed with reference to recent examples. And finally, we focus on insights afforded by IM-MS experiments when applied to the study of conformationally dynamic and intrinsically disordered proteins.