Polyethylene glycol) (PEG) is increasingly used in clinical and experimental medicine. However, quantification of PEG and PEGylated small molecules remains laborious and unsatisfactory. In this report, we stably expressed a functional anti-PEG antibody on the surface of BALB 3T3 cells (3T3/αPEG cells) to develop a competitive enzyme-linked immunosorbent assay (ELISA) for PEG quantification. The αPEG cell-coated plate bound biotinylated PEG5K (CH3-PEG5K-biotin) and CH3-PEG5k- 131I more effectively than did a traditional anti-PEG antibody-coated plate. Competitive binding between PEG (2, 5,10, or 20 kDa) and a known amount of CH3-PEG5K-biotin allowed construction of a reproducible competition curve. The αPEG cellbased competition ELISA measured small molecules derivatized by PEG 2K, PEG5K, PEG10k, PEG20k. and PEG5k at concentrations as low as 58.6,14.6,3.7,3.7, and 14.6 ng/mL, respectively. Notably, the presence of serum or bovine serum albumin enhanced PEG measurement by the αPEG cell-based competition ELISA. Finally, we show here that the αPEG cell-based competition ELISA accurately delineated the pharmacokinetics of PEG5K, comparable to those determined by direct measurement of radioactivity in blood after intravenous injection of CH3- PEG5K- 131I into mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGylated small molecules in vivo.