The purification of PHB-01 plantibody derived from tobacco leaves imposed difficulties when the plantibody solid-liquid extraction design was performed. Thus, our study focused on assessing a combination of an aqueous two-phase extraction (ATPE) procedure and affinity chromatography for solving some of the issues in plantibody purification. This was done using a complete factorial redesign, different polyethylene glycol (PEG)/K2PO4 proportions, and pH values in each partitioning variant.