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Membrane extraction with a sorbent interface (MESI) is a sample preparation technique with a rugged and simple design allowing for solvent-free, on-line performance. When coupled to gas chromatography (GC), MESI is extremely promising for the analysis of volatile organic compounds (VOCs), as it is selective and sensitive enough to detect trace levels of analytes. A new calibration method for the MESI technique is presented herein. The aim of this study was to characterize and quantify the breath biomarker ethylene in human subjects. The dominant calibration method was proven true using standard ethylene to verify changes in mass transfer trends, with variations in flow and temperature. Finally, the dominant calibration method was used to determine ethylene levels in real human breath samples from healthy and smoking volunteers. Results were consistent with those reported in literature. These findings suggest that the dominant calibration technique is a useful tool for monitoring ethylene in human breath.