Reactive oxygen species play a crucial role in cellular processes, but their effects on protein structure and function in vivo remain challenging to study. Here, we used synchrotron-based X-ray footprinting to probe protein structure in live E. coli, using quantitative LC-coupled mass spectrometry of methionine oxidation (MSox) with X-ray dose as a variable. A label-free proteomic analysis identified 2104 proteins from E. coli, with 465 proteins exhibiting MSox modifications distributed across multiple cellular compartments. Changes in MSox modification with increasing X-ray dose revealed a correlation between rates of modification and solvent-accessible surface area in vivo for selected proteins, providing a direct probe of protein structure and its conformational plasticity in the cell.