One of the challenges in producing a PEGylated therapeutic protein is that the PEGylation reaction typically generates a mixture of both singly and multiply PEGylated species. The objective of this study was to examine the feasibility of using ultrafiltration for the purification of a singly PEGylated protein from the multiply PEGylated conjugates. Data were obtained with α-lactalbumin that was PEGylated with a 20 kDa activated PEG, with the ultrafiltration performed over a range of pH and ionic strength using both unmodified and negatively charged composite regenerated cellulose membranes. Purification of the singly PEGylated α-lactalbumin from the multiply PEGylated species was accomplished using a diafiltration process with a negatively charged membrane at pH 5 and an ionic strength of 0.4 mM, conditions that maximized the electrostatic exclusion of the multiply PEGylated species from the charged membrane. The diafiltration process provided more than 97% yield with greater than 20-fold purification between the singly and doubly PEGylated proteins and nearly complete removal of the more heavily PEGylated species. The singly PEGylated α-lactalbumin was recovered as a dilute filtrate solution, although this dilution could be eliminated using a cascade filtration or the final product could be re-concentrated in a second ultrafiltration as part of the final formulation. These results demonstrate the feasibility of using ultrafiltration for the purification of singly PEGylated protein therapeutics. Biotechnol. Bioeng. 2011; 108:822–829. © 2010 Wiley Periodicals, Inc.