BACKGROUND The implementation of efficient processes for the development, recovery and purification of biological products is a main challenge for the bioengineering field. PEGylation can enhance the therapeutic properties of the protein, and often results in heterogeneous population of conjugated species and unmodified protein that presents a protein separations challenge. Non‐chromatography strategies, such as countercurrent distribution in aqueous two‐phase systems (CCD‐ATPS) and ultrafiltration (UF) for the fractionation of PEGylated RNase A conjugates represent attractive alternative worth of consideration. RESULTS In this work the potential application of CCD‐ATPS for the separation of closely related proteins (i.e. mono‐PEGylated and di‐PEGylated RNase A) was performed under selected conditions. Process conditions involved PEG 8000 13% (w/w), phosphate 10.4% (w/w), at pH 7.0 and volume ratio of 1.0 resulted, after 30 transfers in CCD, in distinguishable peaks that can be attributed to the mono‐PEGylated protein (61%) and di‐PEGylated (58%) form of RNase A. Alternatively, an ultrafiltration‐based strategy resulted in the partial separation of mono‐PEGylated and di‐PEGylated protein, 38% and 60%, respectively, in the concentrate with a 30 kDa membrane. Using a 50 kDa membrane the mono‐PEGylated RNase A accumulates in the filtrate (43%) and the di‐PEGylated in the concentrate (45%). CONCLUSIONS The results reported here can be considered for the separation of PEGylated proteins, as a first step in the implementation of a purification process. Copyright © 2012 Society of Chemical Industry