We used a high-resolution mid-IR tunable-laser absorption spectroscopy (TLAS) system with a single IV–VI laser operating near 5.2 μm to measure the level of exhaled nitric oxide (eNO) in human breath. A method of internal calibration using simultaneous eNO and exhaled CO2measurements eliminated the need for system calibration with gas standards. The results observed from internally calibrating the instrument for eNO measurements were compared with measurements of eNO calibrated to gas standards and were found to be similar. Various parameters of the TLAS system for eNO breath testing were examined and include gas cell pressure, exhalation time, and ambient NO concentrations. A reduction in eNO from elevated concentrations (44 parts in 109) to near-normal levels (< 20 parts in 109) from an asthmatic patient was observed after the patient had received treatment with an inhaled glucocorticoid anti-inflammatory medication. Such measurements can help in evaluating airway inflammation and in monitoring the effectiveness of anti-inflammatory therapies.