A new method to examine the structure and stability of proteins using footprinting is applied to examine the unfolding of apomyoglobin. Unlike previous cleavage based footprinting methods, synchrotron X-ray protein footprinting is based on a quantitative determination of the extent and the site of radiolytic modification of amino acid side chains, analyzed using mass spectrometry. The amino acids most susceptible to radiolytic oxidation (cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, proline, and leucine) serve as convenient probes of protein structure to monitor changes in solvent accessibility. To determine if the technique can measure quantitative properties of proteins relevant to structure and function, we examined the equilibrium unfolding of apomyoglobin in urea and compared the results to data derived from fluorescence studies under the same conditions.