The use of monoclonal antibodies (mAbs) in medical treatments and in laboratory techniques has a very important impact in the battle against many diseases, namely in the treatment of cancer, autoimmune diseases and neural disorders. Thus these biopharmaceuticals have become increasingly important, reinforcing the demand for efficient, scalable and cost-effective techniques for providing pure antibodies. Aqueous two-phase systems (ATPS) have shown potential for downstream processing of mAbs. In this work, an ATPS in a microfluidic platform was designed and tested for mAbs extraction. The system demonstrated the potential to be an effective tool to accelerate bioprocess design and optimization. The partition of immunoglobulin G (IgG) tagged with fluorescein isothiocyanate (FITC) in an ATPS of polyethylene-glycol (PEG)/phosphate buffer with NaCl was investigated using a PDMS microfluidic device fabricated using soft lithography techniques. Different structures were tested with different values of microchannel length (3.14–16.8 cm) and flow rates of the salt (1–2 μL/min) and PEG-rich phases (0.2–0.5 μL/min). A stable interphase between the phases was obtained and the phenomena of diffusion and of partition of the IgG from the salt-rich phase to the PEG-rich phase were measured by fluorescence microscopy. Process simulation allowed the modeling of the IgG diffusion and partitioning behavior observed in the microstructure. The reduction to the microscale does not greatly affect the antibody extraction yield when compared with macroscale results, but it does reduce the operation time, demonstrating the potentiality of this approach to process optimization.