Honey containing tylosin was studied to determine the stability of residues over time. Residues were isolated from diluted honey samples by solid-phase extraction followed by quantification using high performance liquid chromatography. Oasis HLB cartridge isolation allowed the concentration of tylosin and desmycosin to be determined separately on the same sample. Oasis MCX extraction gave a single compound (apparently desmycosin dimethyl acetal) from both tylosin and desmycosin, with HPLC peak area proportional to the sum of the two. Tylosin converted to desmycosin with a half-life of approximately 4 months at 34 °C. The desmycosin seemed to be quite stable in honey, and the sum of the two antibiotics, determined by either method, decreased only slightly over 9 months. Use of tylosin for treatment of American foulbrood requires caution to prevent persistent residues in honey.