Abstract <p>Multicomponent analysis of residual veterinary drugs by HPLC–MS/MS is relevant for ensuring food safety. A significant problem is the determination of veterinary drugs with large molecules. Electrospray ionization of such molecules is difficult, and it is difficult to achieve the required sensitivity by classical isolation from the matrix with subsequent detection. Such drugs include avilamycin and nosiheptide. This problem can be solved by the fragmentation of molecules into smaller, more easily detectable residues by hydrolysis, which, in turn, poses the task of optimizing the conditions for such preparation, as well as assessing the uniqueness of the selected marker. A possibility of the simultaneous determination of avilamycin and nosiheptide in chicken meat by their alkaline hydrolysis to markers, dichloroisoverninic acid and 4-hydroxymethyl-3-methylindole-2-carboxylic acid, respectively, is shown. The conditions of extraction, hydrolysis, and methods of further purification are studied. Under the selected conditions, the recovery of analytes was &gt;85%; in addition, satisfactory reproducibility (RSD ≤ 15%) was achieved. The limits of detection were 5 μg/kg. The developed procedure is highly selective, rapid, easy to implement, and inexpensive and has good analytical characteristics.</p>