This work addresses the use of protic ionic liquids (PIL) as adjuvants (5 wt%) in the formation of aqueous two-phase systems (ATPS), based on polyethylene glycol with different molecular weights + ammonium sulfate + water for peroxidase purification from alternatives vegetables as radish peroxidase (Raphanus sativus L) from horseradish root peroxidase. Protic ionic liquid as a process adjuvant did not extend the biphasic area in the phase diagram, but the phase behavior was modified according to the hydro-phobicity of the PIL used and additional interactions between the enzyme and PIL increased the purification factor of the radish peroxidase from approximate to 2.5 (no ionic liquid) to approximate to 19.2 fold using 2-hydroxyethylammonium propionate as adjuvant in ATPS. (c) 2017 Elsevier B.V. All rights reserved.