BACKGROUND Assays identifying circulating tumor cells (CTCs) allow noninvasive and sequential monitoring of the status of primary or metastatic tumors, potentially yielding clinically useful information. However, to the authors' knowledge, the effect of radiation therapy (RT) on CTCs in patients with non–small cell lung cancer (NSCLC) has not been previously explored. METHODS This report describes results from a pilot study of 30 patients with NSCLC who received RT. Peripheral blood samples obtained from these patients were assayed for CTCs using an assay that identified live cells using an adenoviral probe that detected the elevated telomerase activity present in almost all cancer cells, but not in normal cells, and the validity of the assay was confirmed with secondary tumor‐specific markers. Patients were assayed before initiation of RT (pre‐RT), during the RT course, and/or after the completion of RT (post‐RT). RESULTS The assay successfully detected CTCs in the majority of patients, including 65% of patients before the start of RT, and in patients with both epidermal growth factor receptor wild‐type and mutation‐positive tumors. The median CTC counts in patients before RT was 9.1 CTCs per mL (range, undetectable to 571 CTCs per mL) and was significantly higher than the average post‐RT count of 0.6 CTCs per mL (range, undetectable to 1.8 CTCs per mL; P<.001). Sequential CTC counts were available in a subset of patients and demonstrated decreases after RT, except for 1 patient who subsequently developed distant failure. CONCLUSIONS The current pilot data suggest that CTC counts appear to reflect response to RT in patients with localized NSCLC. On the basis of these promising results, the authors have launched a more comprehensive and detailed clinical trial. Cancer 2015;121:139–149. © 2014 American Cancer Society. We describe here results of a pilot study of 30 patients undergoing radiation therapy (RT) for non‐small cell lung cancer (NSCLC), from whom peripheral blood samples were assayed for circulating tumor cell (CTC) via a telomerase‐based assay that identifies live cancer cells in the circulation. These pilot data, which we believe to be the first using this robust assay in patients undergoing RT for localized NSCLC, suggest that CTC counts appear to reflect a response to treatment.