Quantitative pharmacokinetic analysis of methoxy-poly(ethylene glycol) (mPEG) and mPEGylated molecules is important for clinical drug development. Here we developed sensitive sandwich and competitive ELISAs by expressing an anti-mPEG antibody on the surface of fibroblasts for effective capture of mPEG molecules in biological samples. α-mPEG sandwich ELISA could quantify the higher-molecular-weight of mPEG (2, 5, and 20 kDa) and mPEGylated molecules. α-mPEG cell-based competitive ELISA was developed to measure the lower-molecular-weight of mPEG molecules (559, 750, and 1000 Da) at nanomolar levels. In addition, α-mPEG cell-based ELISA was unaffected by the presence of 10% human serum or murine serum. We further demonstrate that the α-mPEG cell-based ELISA determined similar pharmacokinetics of mPEG5K as traditional gamma counting of 131I-mPEG5K. The α-mPEG cell-based ELISA may provide an accurate, high sensitivity and easy-to-use tool for directly measuring mPEG and mPEGylated molecules in complex biological samples to accelerate the clinical development of mPEG drugs.