“Footprinting” refers to assays that examine ligand binding and conformational changes by determining the solvent accessibility of the backbone, bases, or side chain structures of macromolecules through their sensitivity to chemical or enzymatic cleavage or modification reactions. Protein footprinting methods have been developed to examine protein structure and conformational changes by monitoring solvent accessibility using either modification or cleavage reactions. Examples of such methods include acetylation, nonspecific limited proteolysis, or probing the accessibility of a range of sites that can be specifically or nonspecifically cleaved or modified. The basis of these diverse chemical approaches is to monitor the change in accessibility of susceptible residues as a function of relevant conformational fluctuations. Protein footprinting methods based on protease cleavage have been used to map protein structure, nucleic acid-protein interactions, and protein folding intermediates. However, cleavage techniques using proteases suffer from limited structural resolution due to the large size of the probe. Hydroxyl radical methods of cleavage and modification have been pursued to overcome this drawback. In this article, we introduce the methods of footprinting, including hydroxyl radical methods (section 1), review the various approaches to generate hydroxyl radicals (section 2), and review the chemistry of hydroxyl radical mediated oxidation of the protein backbone and side chains (section 3). Although some examples of the use of the method are provided, this is not emphasized, as previous reviews have provided a number of relevant examples. 1-3 Thus, this review provides a comprehensive reference work for understanding the chemistry of radical generation and oxidative reactions appropriate for application to structural mass spectrometry experiments.