Breath testing has potential for the rapid assessment of the source and impact of exposure to air pollutants. During the development of a breath test for acetonitrile using selected ion flow tube mass spectrometry (SIFT-MS) raised acetonitrile concentrations in the breath of volunteers were observed that could not be explained by known sources of exposure. Workplace/laboratory exposure to acetonitrile was proposed since this was common to the volunteers with increased breath concentrations. SIFT-MS measurements of acetonitrile in breath and air were used to confirm that an academic chemistry laboratory was the source of exposure to acetonitrile, and quantify the changes that occurred to exhaled acetonitrile after exposure. High concentrations of acetonitrile were detected in the air of the chemistry laboratory. However, concentrations in the offices were not significantly different across the campus. There was a significant difference in the exhaled acetonitrile concentrations of people who worked in the chemistry laboratories (exposed) and those who did not (non-exposed). SIFT-MS testing of air and breath made it possible to determine that occupational exposure to acetonitrile in the chemistry laboratory was the cause of increased exhaled acetonitrile. Additionally, the sensitivity was adequate to measure the changes to exhaled amounts and found that breath concentrations increased quickly with short exposure and remained increased even after periods of non-exposure. There is potential to add acetonitrile to a suite of VOCs to investigate source and impact of poor air quality.