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Antibiotic extraction from solid matrices is generally complicated unlike aqueous phases. Consequently, increasing the extraction efficiency of antibiotics from solid matrices has received a great concern. Therefore, the main objective of this study was to determine three macrolide antibiotics; tylosin (TYL), spiramycin (SPIR), and erythromycin (ERY) in buffaloes’ meat using the recommended method of thin-layer chromatography-bioautography (TLC-B) after some modifications. Antibiotics were extracted by 0.2% metaphosphoric acid-methanol (6:4, v/v), and then the extracts were cleaned up using an Oasis HLB cartridges (200 mg). The recovery ratios at 0.5, 1, and 2 maximum residue limits (MRLs) with high precision were 84.19-92.22%, 83.09-89.42%, and 84.89-90.28%, respectively. The detection limits were 12, 45, and 2 ng g(-1) for TYL, SPIR, and ERY, respectively. Forty-five samples of Egyptian buffaloes’ meat were analyzed using the adapted method. Only 9 and 5 samples out of the 45 samples were positive for TYL and ERY, respectively, with mean concentrations of 42.59 and 31.21 (ng g(-1)) which were lower than the permitted MRLs recommended by the European community.