In this study, size exclusion high performance liquid chromatography was evaluated for its application in separation and quantitation of free polyethylene glycol (PEG) and its PEGylated-protein-conjugate (PEG-conjugate). Although the large mass of the free PEG (2-fold greater than the protein) made separation difficult, chromatographic conditions were identified enabling resolution and quantitation of the free PEG, PEG-conjugate and non-PEGylated protein with Shodex Protein KW803 and KW804 columns in series and refractive index detection. The optimum resolution of 1.7 and 2.0 was achieved for the free PEG and PEG-conjugate as well as the free PEG and non-PEGylated protein using 20 mM HEPES buffer at pH 6.5. Under this condition, the plot of log10MW of all the pertinent analytes against retention time showed a linear relationship with a correlation coefficient of 1. Limited assay performance evaluation demonstrated that the method was linear in the concentration range of 10 to 250 μg/mL of free PEG with correlation coefficients of ≥0.99. When free PEG in this concentration range was spiked into PEG-conjugate samples at 1 mg/mL, the recovery was in the range of 78%–120%. Detection and quantitation limits were determined to be, respectively, 10 and 25 μg/mL for free PEG. The R.S.D. for intra- and inter-day precision was 0.09% or less for retention time measurements and 2.9% or less for area count measurements. Robustness testing was performed by deliberately deviating ±0.2 pH units away from the desired pH as well as by increasing the flow rate. These deviations resulted in no significant impact on area percent distribution of all species. However, separation was found to be sensitive to high ionic strength and buffer species.