Intensive chicken production leads to overuse of antibiotics on poultry farms. For food safety control, there is great need for means to use non-conventional matrices allowing analysis of antibiotics during poultry breeding. The main goal of this study was to demonstrate feathers as suitable material for non-invasive detection of doxycycline treatment in poultry. Transfer to and depletion of doxycycline in chicken feathers were investigated after therapeutic, spray, and subtherapeutic treatment. For the quantitative determination of doxycycline in feathers, a validated ultra-high-performance liquid chromatography - tandem mass spectrometry method was used. High concentrations of doxycycline in feathers were detectable for 22 D post treatment in each experimental group, and they were much higher than those in muscle and liver. A washing experiment with the same solvent as for extraction showed different ratios between extractable and non-extractable residues in feathers of chickens treated therapeutically, by spraying and subtherapeutically, which demonstrates the ability of feather analysis to distinguish different forms of treatment. After a segmentation procedure, high amounts of doxycycline were found to be deposited in the upper part of feathers in each treatment group. The obtained results showed that chicken feathers are a suitable material for the detection and non-invasive surveillance of doxycycline.