Extraction of rutin from acerola waste was investigated using alcohol-salt-based aqueous two-phase systems (ATPS). Initially, the partitioning was studied using model systems with pure and commercial rutin. The impact of the ATPS constituents and composition, initial amount of rutin, temperature and addition of electrolytes was evaluated. Rutin can be recovered either in the alcohol-or-salt-rich phase depending on the salt used. To validate the optimization process, rutin extraction from acerola waste was carried out further. The results obtained with the real samples are in close agreement with the model systems and validate the optimization tests and support their applicability in bioresource-related processes.