Under the EU official monitoring programs, the antibiotics are controlled in tissues of food producing animals. However, regarding the overuse of medicines in veterinary practice, there is a strong need to find an alternative to post-mortem analysis of antibiotics. The use of feathers, as an unconventional matrix, enable to control of birds treatment during breeding. Thus, a novel ultra-high performance liquid chromatography–tandem mass spectrometry method for the analysis of 53 compounds from 11 groups of antibacterials, including penicillins, cephalosporins, macrolides, tetracyclines, quinolones, sulfonamides, aminoglycosides, pleuromutilins, diaminopirymidynes, diaminopirymidynes derivatives and lincosamides in chicken feathers has been developed. The isolation of analytes by solvent extraction method with oxalic acid, Na2EDTA and acetonitrile was performed. The extracts were cleaned by filtration with OASIS HLB cartridges. Chromatographic separation was achieved on a Zorbax SB-C18 analytical column using mobile phase consisting of 0.025% heptafluorobutyric acid and acetonitrile. All compounds were successfully validated, with good sensitivity, accuracy and precision. The developed method gives an opportunity to effective monitoring, whether chickens have been given antibiotics, as well as let to identify the medicines that were used.