Avilamycin was extracted from feed with acetonitrile. Isolation of avilamycin factors from feed matrix interference was accomplished by normal-phase solid-phase extraction with silica as sorbent. Reversed-phase liquid chromatography was subsequently used to separate and quantitate the primary biologically active factors A and B for determination of chemical potency. This method combines specificity for avilamycins A and B in poultry feeds with simple sample preparation that removes matrix interferences. Recoveries of factor A ranged from 93.29 to 97.26%, with precision (relative standard deviation) ranging from 1.1 to 3.4%. Avilamycin factors in feed samples tested ranged from 4.45 to 17.82 micrograms/g for factor A and from 0.80 to 3.18 micrograms/g for factor B.