A determination method of nosiheptide in formula feeds by HPLC-FL was developed and validated, including an inter-laboratory study. Formula feeds were extracted with acetone after adding acetic acid. Liquid chromatographic separation was performed using a ZORBAX Eclipse XDB-C18 column, with acetonitrile and water containing acetic acid as the mobile phase. Detection of NH was carried out with a fluorescence detector. Recovery tests and an inter-laboratory study were conducted using chicken and swine formula feeds fortified with nosiheptide at 0.5-27 mg-potency/kg. Mean recoveries in recovery tests ranged from 91.4 to 103%, and the repeatability in terms of relative standard deviation was within 7.8%. Mean recoveries of the inter-laboratory study ranged from 98.4 to 108%, the repeatability and reproducibility in terms of the relative standard deviations were within 8.1% and 13% respectively, and the HorRat values ranged from 0.21 to 0.75.