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This Article describes a density-based method for removing contaminants, including microorganisms and nonviable cells, from mammalian cell cultures using an aqueous two-phase system (ATPS). The properties of a 7% w/w polyethylene glycol (PEG)-11% w/w Ficoll ATPS can be tuned to prepare a biocompatible system that removes contaminants with little to no adverse effects on the viability or growth of the cultured cells after treatment. This system can be used to enrich cell culture populations for viable cells and to reduce the number of microorganism contaminants in a culture, which increases the chances of subsequent antibiotic treatments being successful. We test the effectiveness of our method in model contaminated cultures of both adherent (HeLa) and suspension (HL-60 II) mammalian cells contaminated with bacteria (E. coli) and yeast (S. cerevisiae). An average of 70.2 ± 4.6% of HeLa cells added to the system are subsequently recovered, and 55.9 ± 2.1% of HL-60 II cells are recovered. After sedimenting to the interface of the ATPS, these cells have an average viability of 98.0 ± 0.2% and 95.3 ± 2.2%, respectively. By removing unwanted cells, desired cell populations can be recovered, and cultures that would otherwise need to be discarded can continue to be used.