Aqueous two-phase systems (ATPS) represent a promising strategy for the recovery of CD133(+) stem cells. This particular type of stem cells has great potential for research and clinical applications. Traditional [polyethylene glycol (PEG), dextran (DEX), and ficoll] and novel (Ucon) polymer-polymer ATPS were exploited to study the partitioning behavior of CD133(+) stem cells and contaminants from human umbilical cord blood (HUCB). The aim of the study was to select conditions under which the product of interest and the contaminants concentrate in opposite phases. To accomplish this, three independent samples were tested: (1) enriched CD133(+) sample, (2) whole HUCB (contaminants), and (3) complex sample (CD133(+) stem cells and contaminants). The objective of this research was to evaluate the partition behavior of CD133(+) in ATPS in route to establish the basis for the development of a novel and scalable purification bioprocess. In conclusion, the partitioning behavior of CD133(+) stem cells and contaminants from complex samples was as follows: 59% of CD133(+) stem cells fractionated to the top phase when employing ficoll 400,000-DEX 70,000 or 100% to the bottom phase with Ucon-DEX 75,000 and PEG 8,000-DEX 500,000 ATPS. In average, 35% of the contaminants partitioned to the top phase of the ficoll 400,000-DEX 70,000 ATPS, 99% to the dextran rich phase of the Ucon-DEX 75,000 systems and 97% to the bottom phase of the PEG 8,000-DEX 500,000. Cell viability was at least 98% after ATPS recovery.