Cetuximab is an anti-epidermal growth factor receptor monoclonal antibody used in the treatment of colorectal and head and neck cancers. Part of the interindividual differences in response may be explained by interindividual variability in pharmacokinetics. An assay measuring cetuximab serum concentrations is therefore needed. An enzyme-linked immunosorbent assay was developed using microtiter plates sensitized with a recombinant human epidermal growth factor receptor extracellular domain. Lower and upper limits of quantitation and limit of detection were determined. Eight standard calibrators (SCs) and 3 quality controls (QCs), that is, 0.75, 7.5, and 15 mg/L, were tested on 5 occasions on 1 day and on 5 occasions on different days. Trough and peak serum concentrations of cetuximab were measured in 15 patients with metastatic colorectal cancer and 1 patient with undetermined neoplasia undergoing cetuximab-based chemotherapy. Cetuximab concentrations were described using a 2-compartment population pharmacokinetic model. Imprecision and accuracy of SC and QC were < or = 20%, except for the 0 and 0.1 mg/L SC concentrations (< or = 20%). The limit of detection was 0.012 mg/L. Lower and upper limits of quantitation were 0.75 and 15 mg/L, respectively. A total number of 198 blood samples were available from the 16 patients. Median (range) trough and peak concentrations during the treatment were 49.6 (5.8-105.4) and 177.2 (97.5-235) mg/L, respectively. This method is rapid, accurate, and reproducible and may be useful for pharmacokinetic and pharmacokinetic-pharmacodynamic studies, as well as in therapeutic drug monitoring of cetuximab.