Reversed-phase liquid chromatography (RPLC) of intact proteins induces conformational changes due to the denaturing effects of both the hydrophobic stationary phase and the high-organic mobile phase, which can lead to poor recovery and badly distorted or artifactual chromatographic peaks. A surprising additional consequence is that the length of the column does not matter. We are investigating these structural changes using mass spectrometric protein footprinting, which gives information about changes in the solvent accessibility of modifiable sites. Differences in site accessibility following adsorption to and desorption from chromatographic surfaces indicate regions of structural alteration in proteins. We seek to determine if bovine serum albumin (BSA) adsorbs in a preferred binding orientation, if denaturation increases with surface residence time, and if the conformation in high-organic solvent depends on whether the protein was first adsorbed. Glutamate and aspartate groups of BSA are amidated with methylamine and EDAC. Tryptic digests of the modified proteins are analyzed using LC-MS/MS. Peptides are identified with tandem mass spectrometry, and modification sites are located through mass shifts in the tandem mass spectra. Quantification is performed by comparing relative peak areas of modified and unmodified peptides in extracted ion chromatograms for their respective precursors. While results remain somewhat ambiguous in the absence of instrumental replicates of individual samples and modeling, our research presents a method for footprinting proteins adsorbed to chromatographic surfaces and confirms the apparent contribution of both the hydrophobic stationary phase and the high-organic mobile phase to conformational changes observed during RPLC.