Detection of macrolides residues in animal feedingstuffs to ensure food safety is very important issue. For this purpose, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of residues of 4 macrolides (erythromycin, spiramycin, tilmicosin and tylosin) in animal feedingstuffs. Roxithromycin (ROX), a macrolide not used in veterinary medicine, was used as internal standard. The mass spectral acquisition was done in the positive-ion mode applying multiple reaction monitoring with the following ions (mass-to-charge ratio, m/z): m/z 837.4 -> 679.2; m/z 734.4 -> 576.2; m/z 422.4 -> 174.1; m/z 869.1 -> 696.1 and m/z 916.4 -> 772.4 for roxithromycin (ROX), erythromycin (ERY), spiramycin (SPI), tilmicosin (TIL), and tylosin (TYL), respectively. Good repeatibility, reproducibility and recovery values was obtained. Average recoveries ranged from 98.9% (TIL) to 101% (ERY) and an overall mean of 99.9%. Method limits of detection (LODs) of ERY, SPI, TIL and TYL 5.1, 6.6, 6.7 and 7.5 mu g kg-1 were achieved respectively. Method limits of quantification (LOQs) were 8.5, 11.1, 11.2 and 12.4 mu g kg-1 for the same drugs, respectively. Satisfactory decision limits (CC alpha) and detection capabilities (CC beta) were also attained. The method is simple, rapid, sensitive and suitable for the simultaneous determination of macrolide antibiotics in animal feedingstuffs.