Mass spectrometry (MS)-based proteomics methods can give unique insight into protein structure and interactions. Here, we have developed a novel top-down footprinting method, Methionine Oxidation Footprinting in Intact Proteins (MOFIP), to probe solvent accessibility in intact proteoforms. For MOFIP, natively folded protein lysates are incubated with and without hydrogen peroxide (H2O2) to evaluate solvent accessibility of methionine residues. Top-down proteomics analysis allows the characterization of the solvent accessibility of each methionine residue within intact proteins to obtain structural information. Here, intact E. coli lysate was used to evaluate the feasibility of complex biological sample analysis using the MOFIP platform. Overall, we profiled 137 methionine residues from 69 proteoforms that contained at least one methionine residue suitable for methionine footprinting. These methionine residues from intact proteoforms fell into three general categories: solvent-accessible (50%), solvent-inaccessible (16%), and partially solvent-accessible (34%). Overall, we have demonstrated that our novel MOFIP platform is capable of characterizing the solvent accessibility of individual methionine residues within intact proteoforms in complex biological samples, which is exceedingly challenging using bottom-up proteomics methods.