The authorisation of tylosin as feed additive was withdrawn for reasons of human health concerning resistance of pathogenic bacteria. An analytical method for the identification and quantification of tylosin in animal feed was developed and validated. The samples were extracted using an acidified methanol:water mixture and solid-phase extraction was employed for the isolation of the antibiotic from diluted feed samples. Tylosin was analysed by liquid chromatography with electrospray ionisation mass spectrometric detection. The method’s performance was evaluated in adherence to the Commission Decision 2002/657/EC. The recovery of the analyte from spiked samples was determined to be in the range from 78.9% to 108.3% depending on tylosin concentrations. The CCα and CCβ values for tylosin in feeds were determined at 0.085 mg kg-1 and 0.091 mg kg-1, respectively. The method detection limit was found to be 0.035 mg kg-1 and the quantification limit 0.05 mg kg-1. The applicability of the developed method was tested by analysing real feed samples. A reliable LC-MS method was developed to identify and quantify tylosin in animal feed with a good repeatability and a high specificity and sensitivity. Because of these characteristics, the proposed method is applicable and could be deemed necessary within the field of feed control and safety.