Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron “white” beam results in the formation of stable side-chain oxidation products, which can be digested with proteases for mass spectrometry analysis. LC-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side-chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side-chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its “inactive” and Ca+2- activated state. Oxidation rate data for eighty-one peptides derived from the trypsin digestion of gelsolin are presented; sixty of these peptides were observed not to be oxidized and twenty-one had detectible oxidation rates. We also report the Ca+2-dependent changes in oxidation for all eighty-one peptides; fifty-nine remain unoxidized, while five increase their oxidation rate and two experience protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca-insensitive and Ca-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand induced structural changes and the data provide a detailed model for gelsolin activation.