Protein higher-order structure (HOS) is key to biological function because the mechanisms of protein machinery are encoded in protein three-dimensional structures. Mass spectrometry (MS)-based protein footprinting is advancing protein structure characterization by mapping solvent-accessible regions of proteins and changes in H-bonding, thereby providing higher order structural information. Footprinting provides insights into protein dynamics, conformational changes, and interactions, and when conducted in a differential way, can readily reveal those regions that undergo conformational change in response to perturbations such as ligand binding, mutation, thermal stress, or aggregation. Building on firsthand experience in developing and applying protein footprinting, we provide an account of our progress in method development and applications.In the development section, we describe fast footprinting with reactive reagents (free radicals, carbenes, carbocations) with emphasis on fast photochemical oxidation of proteins (FPOP). The rates of the modifying reactions are usually faster than protein folding/unfolding, ensuring that the chemistry captures the change without biasing the structural information. We then describe slow, specific side-chain labeling or slow footprinting and hydrogen-deuterium exchange (HDX) to provide context for fast footprinting and to show that, with validation, these modifications can deliver valid structural information. One advantage of slow footprinting is that usually no special apparatus (e.g., laser, synchrotron) is needed. We acknowledge that no single footprint is sufficient, and complementary approaches are needed for structure comparisons.In the second part, we cover several of our footprinting applications for the study of biotherapeutics, metal-bound proteins, aggregating (amyloid) proteins, and integral membrane proteins (IMPs). Solving structural problems in these four areas is often challenging for other high-resolution approaches, motivating the development of protein footprinting as a complementary approach. For example, obtaining structural data for the bound and unbound forms of a protein requires that both forms are amenable for 3D structure determination. For problems of this type, information on changes in structure often provides an answer. For amyloid proteins, structures of the starting state (monomer) and the final fibril state are obtainable by standard methods, but the important structures causing disease appear to be those of soluble oligomers that are beyond high-resolution approaches because the mix of structures is polydisperse in number and size. Moreover, the relevant structures are those that occur in cell or in vivo, not in vitro, ruling out many current methods that are not up to the demands of working in complex milieu. IMPs are another appropriate target because they are unstable in water (in the absence of membranes, detergents) and may not retain their HOS during the long signal averaging needed for standard tools. Furthermore, the structural changes occurring in membrane transport or induced by drug binding or other interactions, for example, resist high resolution determination.We provide here an account on MS-based footprinting, broadly describing its multifaceted development, applications, and challenges based on our first-hand experience in fast and slow footprinting and in HDX. The Account is intended for investigators contemplating the use of these tools. We hope to catalyze refinements in methods and applications through collaborative, cross-disciplinary research that involves organic and analytical chemists, material scientists, and structural biologists.