An upstream process was developed for large-scale assembly of bispecific F(ab')(2) from two different Fab' molecules. We identified a novel product-related structural variant that is difficult to remove downstream and modified our process to decrease its generation during the assembly step. The resulting multistep process includes affinity capture, an on-column cysteine uncapping step, batch reoxidation and pyridylation, and assembly of the two Fab' molecules into a F(ab')(2). Optimizations include selection of hinge domain-specific reducing agent, column chromatography conditions, and reaction pH. We selected L-reduced Glutathione to target hinge cysteines for reduction, uncapping the Fab' while minimizing off-target reduction. We scaled up the improved method for assembling bispecific F(ab')(2) with similar assembly efficiency but significantly improved product quality, generating a pool with bispecific F(ab')(2) for downstream processing.