Selected volatile organic compounds (VOCs) in breath may be considered biomarkers if they are indicative of distinct diseases or disease states. Given the inherent molecular selectivity of vibrational spectroscopy, infrared sensing technologies appear ideally suitable for the determination of endogenous VOCs in breath. The aim of this study was to determine that mid-infrared (MIR; 3-20 µm) gas phase sensing is capable of determining isoprene in exhaled breath as an exemplary medically relevant VOC by hyphenating novel substrate-integrated hollow waveguides (iHWG) with a likewise miniaturized preconcentration system. A compact preconcentrator column for sampling isoprene from exhaled breath was coupled to an iHWG serving simultaneously as highly miniaturized gas cell and light conduit in combination with a compact Fourier transform infrared spectrometer. A gas mixing system enabled extensive system calibration using isoprene standards. After system optimization, a calibration function obtaining a limit of quantification of 106 ppb was achieved. According to the literature, the obtained sensitivity is sufficient for quantifying middle to high isoprene concentrations occurring in exhaled breath. Finally, a volunteer breath sample was analysed proving comparable values of isoprene in a real-world scenario. Despite its fundamental utility, the proposed methodology contains some limitations in terms of sensitivity and temporal resolution in comparison with the readily available measurement techniques that should be addressed during future optimization of the system. Nonetheless, this study presents the first determination of endogenous VOCs in breath via advanced hollow waveguide MIR sensor technology, clearly demonstrating its potential for the analysis of volatile biomarkers in exhaled breath.