BACKGROUND: A short processing time and efficient scale-up stem cell isolation bioprocess is essential to exploit the potential of these cells for the treatment of multiple chronic diseases. Various methodologies have been used for stem cell recovery. However, most of them present economic and/or time-consuming drawbacks. RESULTS: This paper reports the characterization and optimization of immunoaffinity aqueous two-phase systems (ATPS), a liquid-liquid based separation technology enhanced with the PEGylation of the antibody as a promising platform in route tothe separation of CD133 + stem cells. The best results were obtained with Ucon-DEX or PEG-DEX systems with tie-line length (TLL) 15 or 20% w/w and volume ratio (V R ) 3. When the PEGylated antibodies were added to these selected optimized ATPS, they partitioned to both phases, but with higher preference for the bottom phase. CONCLUSION: PEGylation was an effective strategy to induce a change in the partition behavior of the CD133/2-biotin antibody when Ucon-DEX ATPS were used. Similarly, variation of the parameters (TLL and V R ) resulted to be useful for this goal.Nevertheless, further studies are required to find the optimal ATPS composition that will fractionate 100% of the antibody to the contaminants' opposite phase, making this system an ideal candidate to be tested for the selectivity of CD133 + stem cells. Immunoaffinity ATPS optimization strategyA multilevel categorical design was carried out using Statgraph-ics18 software (Statgraphics Technologies, Inc., The Plains, VA, USA). A total of 27 combinations by duplicate of three TLLs (15, 20 and 25% w/w) and three V R s (0.33, 1 and 3) were explored J Chem Technol Biotechnol 2020; 95: 123-131