Tylosin is one of the most commonly used antimicrobial drugs from the macrolide family and in broiler chickens it is used specially for the treatment of infectious pathologies. The poultry industry produces several by-products, among which feathers account for up to 7% of a chicken's live weight, thus they amount to a substantial mass across the whole industry. Feathers have been repurposed as an animal feed ingredient by making them feather meal. Therefore, the presence of high concentrations of residues from antimicrobial drugs in feathers might pose a risk to global public health, due to re-entry of these residues into the food chain. This work aimed to characterise the depletion behaviour of tylosin in feather samples, while considering its depletion in muscle and liver tissue samples as a reference point. To achieve this goal, we have implemented and validated an analytical methodology suitable for detecting and quantifying tylosin in these matrices. Sixty broiler chickens, raised under controlled conditions, received an oral dose of 32 mg kg of tylosin for 5 days. Tylosin was quantified in muscle, liver and feathers by liquid chromatography coupled with a photodiode array detector (HPLC-DAD). High concentrations of tylosin were detected in feather samples over the whole experimental period after completing both the therapy and the recommended withdrawal time (WDT). On the other hand, tylosin concentrations in muscle and liver tissue samples fell below the limit of detection of this method on the first sampling day. Our results indicate that the WDT for feather samples is 27 days, hence using feather meal for the formulation of animal diets or for other agricultural purposes could contaminate with antimicrobial residues either other livestock species or the environment. In consequence, we recommend monitoring this matrix when birds have been treated with tylosin, within the context of poultry farming.