Avilamycin residue in food is regulated as its marker residue dichloroisoeverninic acid (DIA). An isotope dilution liquid chromatography-tandem mass spectrometry method is established for the accurate determination of DIA in animal muscles without any pre-extraction and preconcentration prior to alkaline hydrolysis. Optimization of the sample cleanup procedures such as liquid-liquid extraction and solid phase extraction was performed by fine-tuning several critical parameters to reduce the matrix effects. Quantification of DIA in edible muscle was accomplished by using matrix-matched calibration with dichloroisoeverninic acid-d6 as internal standard. The method was validated with DIA and avilamycin-fortified poultry and porcine muscles at three different levels (25, 50, and 100 μg/kg). Conversion of avilamycin to DIA by alkaline hydrolysis was ≥92%. The recoveries of DIA in both muscles at three fortification levels ranged from 94 to 106% and RSDs were ≤11% in all cases. The estimated limit of detection values in poultry and porcine muscles were 2.7 and 0.7 μg/kg, respectively. The estimated limit of quantitation values in poultry and porcine muscles were 8.3 and 2.4 μg/kg, respectively. This method is suitable for routine monitoring of avilamycin residue in food safety surveillance programs.