The use of limited proteolysis followed by peptide mapping for the comparability of the higher-order structure of biopharmaceuticals was investigated. In this approach the proteolysis is performed under non-reducing and non-denaturing conditions, and the resulting peptide map is determined by the samples primary and higher order structures. This allows comparability of biopharmaceuticals to be made in terms of their higher order structure, using a method that is relatively simple to implement. The digestion of a monoclonal antibody under non-denaturing conditions was analyzed using peptide mapping, circular dichroism (CD) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This allowed an optimal digestion time to be chosen. This method was then assessed for its ability to detect structural change using a monoclonal antibody, which had been subjected to a range of stresses; deglycosylation, mild denaturation and a batch that had failed specifications due to in-process reduction. The repeatability and inter-assay precision were assessed. It was demonstrated that the limited proteolysis peptide maps of the three stressed samples were significantly different to control samples and that the differences observed were consistent between the occasions when the assays were run. A combination of limited proteolysis and CD or SDS-PAGE analysis was shown to enhance the capacity of these techniques to detect structural change, which otherwise would not have been observed.