The fetal oncogene 5T4 is a cell surface protein, with overexpression observed in a variety of cancers as compared to normal adult tissue. The ability to select patients with tumors that express high levels of 5T4 may enrich a clinical trial cohort with patients most likely to respond to 5T4 targeted therapy. To that end, we developed assays to measure 5T4 in both tumors and in circulating tumor cells (CTCs). We identified the presence of 5T4 in both adenocarcinoma and squamous cell carcinoma of lung, in all clinical stages and grades of disease. CTCs were identified in peripheral blood from the majority of patients with NSCLC, and 5T4 was detectable in most samples. Although 5T4 was present in both CTCs and tumors in most patients, there was no concordance between relative amount in either sample type. Clinical response rates of patients treated with the therapies directed against 5T4 in early stage clinical trials, as determined by these assays, may provide important insights into the biology of 5T4 in tumors and the mechanisms of action of 5T4-targeting therapy.