Abamectin, doramectin, moxidectin, ivermectin, milbemectin A3 and milbemectin A4 are similar macrocyclic lactone chemicals used as parasiticides or acaricides. A method using high performance liquid chromatography (HPLC) with fluorescence detection is presented for the simultaneous determination of the residue amounts of these compounds in bovine muscle. Samples are extracted using acetonitrile and cleaned up with solid phase extraction using a C18 column, followed by fluorescence-derivatized with 1-methylimidazole and trifluoroacetic anhydride in acetonitrile. The analogue was measured by HPLC with fluorescence detector at 365 nm excitation and 470 nm emission wavelengths. The limits of quantification are below the stipulated Taiwan Maximum Residue Limit for each compound. The recoveries of this method in bovine muscle ranged from 73.3 to 110%, with a RSD from 2.11 to 16.57%. The detection limit of those 6 compounds in bovine muscle was 5 ppb. No any above compounds were detected in 50 samples of bovine muscle tested. Therefore, the developed method can be used for rapid screen of macrocyclic lactones in bovine muscle.