Abstract: The sample of beef tendon was extracted with acetonitrile, and the extract obtained was purified by passing through C18 SPE column. The purified solution was derivatized with N-methylimidazole and trifluoroacetic anhydride (dissolved in acetonitrile) at room temperature. The derivatized product of avermectin (AVM) and ivermectin (IVM) was used for HPLC determination with fluorescence detection using C18 chromatographic column (4.6 mm×150 mm, 5 μm) as stationary phase and mixtures of acetonitrile and H2O(97+3) as mobile phase. Linear relationships were found between values of fluorescence intensity and mass fraction of AVM in the range of 5-100 ng·g-1 and IVM 10-100 ng·g-1 as measured at wavelengths of 365 nm (λex) and 470 nm (λem). Values of detection limit (3S/N) of AVM and IVM found were 2.5 ng·g-1 and 4.0 ng·g-1 respectively. Tests for recovery were performed by standard addition method at 3 concentration levels for 6 parallel determinations; results of recovery found were in the range of 81.6%-101.0% for AVM and 101.5%-102.4% for IVM, with RSD′s ≤2.0%.