Cathepsin D has been identified as a challenge to remove in downstream bioprocessing of monoclonal antibodies (mAbs) due to interactions with some mAbs. This study focused on investigating the mechanisms of interaction between cathepsin D and two industrial mAbs using a combined experimental and computational approach. Surface plasmon resonance was used to study the impact of pH and salt concentration on these protein-protein interactions. While salt had a moderate effect on the interactions with one of the mAbs, the other mAb demonstrated highly salt-dependent association behavior. Cathepsin D binding to the mAbs was also seen to be highly pH dependent, with operation at pH 9 resulting in a significant decrease in the binding affinity. Protein-protein docking simulations identified three interaction sites on both mAbs; near the complementarity determining region (CDR), in the hinge, and in the C(H)3 domain. In contrast, only one face of cathepsin D was identified to interact with all the three sites on the mAbs. Surface property analysis revealed that the binding regions on the mAbs contained strong hydrophobic clusters and were predominantly negatively charged. In contrast, the binding site on cathepsin D was determined to be highly positively charged and hydrophobic, indicating that these protein-protein interactions were likely due to a combination of hydrophobic and electrostatic interactions. Finally, covalent crosslinking coupled with mass spectrometry was used to validate the docking predictions and to further investigate the regions of interaction involved in mAb-cathepsin D binding. A strong agreement was observed between the two approaches, and the CDR loops were identified to be important for cathepsin D interactions. This study establishes a combined experimental and computational platform that can be used to probe mAb-host cell protein (HCP) interactions of importance in biomanufacturing.