The presence of antibiotic residues in the food chain may pose a serious risk to human health. Locating and evaluating new sources of consumer exposure to antibiotic residues in food is a very important element of health protection. The possibility of doxycycline uptake from the substrate for mushroom cultivation by the white button mushroom (Agaricus bisporus) fruit body was investigated. Mushrooms were experimentally cultivated on substrate contaminated with 8 different doxycycline concentrations in substrate and analyte concentrations in mushroom samples were measured using ultra-high performance liquid chromatography – triple quadrupole tandem mass spectrometry (UHPLC–MS/MS) The obtained results clearly indicated that doxycycline transfers from contaminated substrate to mushrooms at concentrations ranging from 0.87 to 72.3 µg/kg, depending on substrate contamination concentration level and order of harvesting.