Human breath contains hundreds of trace volatile organic compounds. These volatile substances may be generated in the body or may be absorbed as contaminants from the environment. Some of the endogenous substances are characteristic markers of pathophysiological processes and clinicians are already using breath testing of such substances as an additional non-invasive diagnostic tool for certain diseases. Isoprene, another volatile compound, is formed endogenously in humans, and while the biochemical pathways of biosynthesis and exact origins of isoprene found in human breath have not been elucidated in sufficient depth, its measurement in exhaled breath has been suggested as a non-invasive indicator with diagnostic potential. This test has not yet reached the level of routine clinical methods and is still under development. Breath isoprene levels have been reported to be altered in a number of clinical conditions; however, the physiological meaning of these changes has not been established. Various lines of supportive evidence suggest that isoprene is related to cholesterol biosynthesis. Therefore, breath isoprene measurements could potentially be used for mass screening for lipid disorders and could, at minimum, serve as an additional parameter to complement invasive tests for monitoring the efficacy of lipid-lowering therapy, pharmacological and dietary or lifestyle. As a potentially useful biomarker of mevalonate synthesis in humans, it may have non-invasive applications, not only in metabolic disorders, but possibly also in cancer screening. In the present work, we aim to give a concise overview of breath isoprene, its measurement techniques, problems concerning its physiological meaning, and potential applications as a non-invasive biomarker in modern medicine.