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Poly(ethylene 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 and-PEG antibody on the surface of BALLS 3T3 cells (3T3/[alpha]PEG cells) to develop a competitive enzyme-linked immunoserbent assay (ELISA) for PEG quantification. The [alpha]PEG cell-coated plate bound biotinylated [PEG.sub.5K] (C[H.sub.3]-[PEG.sub.5K]-biotin) and C[H.sub.3]-[PEG.sub.5K]-[sup.131]I 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 C[H.sub.3][PEG.sub.5k]-biotin allowed construction of a reproducible competition curve. The [alpha]PEG cellbased competition ELISA measured small molecules derivatized by [PEG.sub.2K], [PEG.sub.5K], [PEG.sub.10K], [PEG.sub.20K], and [PEG.sub.5K] 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 [alpha]PEG cell-based competition ELISA. Finally, we show here that the [alpha]PEG cell-based competition ELISA accurately delineated the pharmacokinetics of [PEG.sub.5K], comparable to those determined by direct measurement of radioactivity in blood meter intravenous injection of C[H.sub.3]-[PEG.sub.5K]-[sup.131]I into mice. This quantitative strategy may provide a simple and sensitive method for quantifying PEG and PEGytated small molecules in vivo. 10.1021/ac902548m