Therapeutic monoclonal antibodies (mAbs) are inherently heterogeneous and hence generally studied and controlled by an array of orthogonal separation methods. During drug candidate development, fractionation by HPLC is regularly employed to assist peak identification and product understanding. One overlooked challenge is the protein oxidation introduced by the fractionation process. In this study, we report the extent of experimentally introduced protein oxidation, which tends to complicate data interpretation and peak assignments. Higher-energy detectors such as fluorescence detectors, lower fraction concentration, and lower salt concentration in elution solvent were found to exacerbate the oxidation artifacts. The buffer exchange and concentration steps after fraction collection were also found to contribute significantly to protein oxidation. Furthermore, our study showed that collecting fractions into a solution with protecting reagents, such as histidine and methionine, was effective in eliminating the oxidation artifacts introduced by detector exposure and fraction processing steps. Through an example, we demonstrate that the modified fractionation workflow described here improves the accuracy of peak assignments.