Abstract: A portion (2.00 g) of homogenized pulp of sample of aquatic product was taken and after addition of 20 μL of 100 μg·L^{-1 13}C₄-tiamulin methanol solution as internal standard, the mixture was extracted with 10 mL of a mixture of formic acid and acetonitrile (2+98) to transfer retapamulin into the extraction solvent through the operations as follows:1 mix by swirling for 30 s; 2 treat ultrasonically for 10 min at 40℃ water bath; and 3 centrifuge for 5 min. An aliquot of 4.5 mL of the supernatant was taken and diluted to 15.0 mL with water. This solution was then purified by passing through Oasis HLB SPE column, and the SPE column was rinsed with methanol-water (5+95) solution; the solution remained on the column was expelled by suction, and then the analyte on the column was eluted with 4 mL of methanol. The eluate was collected and evaporated to dryness by N₂-blowing at 50℃ water bath. 1 mL of the mixture of mobile phases (A) and (B) mixed in the ratio of 80 to 20 was added to dissolve the residue, and the solution was filtered through 0.22 μm filtering membrane. An aliquot of 10 μL of the filtrate was introduced to UHPLC-MS/MS to be separated by using Waters ACQUITY UPLC BEH C₁₈ chromatographic column as stationary phase, and mixtures of 5 mmol·L^-1 NH₄OAc solution (containing 0.5 mL of formic acid in 1 L of solution) (A) and acetonitrile (B), mixed in various ratios were used as mobile phases in the programmed gradient elution. ESI in the mode of positive ion scanning and mode of MRM were adopted in MS/MS. Linearity range for retapamulin was found between 1.0-20.0 μg·L^-1, with detection limit (3S/N) of 0.1 μg·kg^-1. Using 3 aquatic product sample as matrixes, test for recovery was performed by standard addition method giving values of recovery in the range of 98.9% to 105%, and values of RSDs (n=6) in the range of 2.0%-3.8%.