<p>Airborne exposure to mycotoxins and fungal spores poses a significant yet often overlooked risk to human health. Inhalation and ingestion of mycotoxins being adsorbed onto dust particles or as part of mycelium debris are the primary pathways of incorporation. Exposure assessment, however, is hampered as the detection of airborne mycotoxins remains challenging due to very low sampling masses, ultra-trace level toxin concentrations, and the complexity of dust matrices. Here, we present a reliable sampling and LC–MS/MS-based quantification method for 29 mycotoxins highly relevant to airborne mycotoxin exposure. Total suspended particulate matter (TSP) was collected using a stationary sampler equipped with glass fiber filters, which were subsequently classified according to composition and extent of their loading. Extraction conditions were optimized and standard addition applied to match the observed matrix effects. The method was fully validated by&#xa0;applying a paired observations approach covering working ranges and quantification limits applicable to ultra-trace analysis. Applicability of the method has been demonstrated in three authentic cases of mold-infested working environments, where seven different mycotoxins including alternariol monomethyl ether, sterigmatocystin, and stachybotrylactam were successfully detected and quantified.</p> Graphical abstract <p></p>