Abstract Tylosin is a medium-spectrum macrolide antibiotic used exclusively in veterinary medicine for the treatment of a wide range of infections. The purpose of this study was to develop and validate a method to determine tylosin A residues in edible tissues of swine. Extraction of tylosin A from the tissues was performed using liquid extraction with methanol, followed by a solid-phase clean-up procedure on an SCX strong cation-exchange column. Spiramycin was used as the internal standard. After drying of the eluate, the residue was redissolved and further analyzed by reversed phase liquid chromatography–electrospray ionisation–mass spectrometry/mass spectrometry (LC–ESI–MS/MS). Chromatographic separation of the internal standard spiramycin I and tylosin A was achieved on a Nucleosil (5 μm) column using a mixture of 0.01 M ammonium acetate pH 3.5 in water and acetonitrile as the mobile phase. The tylosin A component could be identified from MS/MS detection, and subsequently quantified. The method has been validated according to the requirements of the EC at the maximum residue limit (MRL), 100 ng tylosin A/ g for muscle, skin+fat, kidney and liver), half the MRL and double the MRL levels. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested ( r>0.99 and goodness of fit <10%). Limits of quantification of 5.0 ng g −1 were obtained for the determination of tylosin A in all tissues, which is well below half the MRL. Limits of detection ranged between 0.2 and 0.8 ng g −1 for the various tissues. The within-day and between-day precisions and the accuracy fell within the ranges specified. The method has been successfully used for the quantitation of tylosin A in tissue samples of swine medicated with tylosin via intramuscular injection.