Two parental monoclonal antibodies that have been designed with orthogonal Fab interfaces are co-expressed to orthogonal Fab generation. These interfaces are carefully built based on the X-ray crystal structures of the parental antibodies, which are obtained by computational approaches. By generating mutations at the heavy chain-light chain interface, the design method hopes to increase each light chain's affinity and specificity for its cognate heavy chain while decreasing these attributes for the noncognate heavy chain. Techniques like surface plasmon resonance (SPR), analytical ultracentrifugation (AUC), size-exclusion chromatography (SEC), and electrospray ionization mass spectrometry (ESI-MS) are used in the experimental validation of the orthogonal Fab interfaces.