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An experiment was conducted to examine the feasibility of detecting unique volatile organic biomarkers exhaled by healthy and morbid heifer calves on-site using noninvasive sampling with solid phase microextraction (SPME) and analysis with gas chromatography-mass spectrometry (GC-MS). Two heifers were selected on the bases of health status and were sampled from 5 to 15 min with a special facemask sampling system using DVB/Carboxen/PDMS 50/30 m and PDMS 100 m SPME fibers. The samples were sealed and transported to the laboratory and analyzed with ion trap GC-MS. Acetone, methyl ethyl ketone, toluene, pentadecane, and nonanal were detected in cattle breath. Tetradecane and decanal were detected in the breath of the sick heifer only. Detector responses to breath VOCs were generally higher for the morbid heifer than the healthy one. Longer extraction time (15 min) resulted in larger detector responses and greater number of compounds detected compared to the short sampling time (5 min). A simple sampling system, combined with SPME-based sampling and analysis with GC-MS was very useful for the detection of bovine breath and ruminal gases. DVB/Carboxen/PDMS 50/30 m coating, Teflon caps, and sample refrigeration worked well for sample preservation and retention of compounds found in breath samples. SPMEbased sampling of animal breath has the potential of being used as a rapid, non-invasive on-site diagnostic and research tool for cattle producers, veterinarians, and researchers.