Afoxolaner is an insecticide and acaricide that belongs to the isoxazoline chemical compound group. it has been used as an active pharmaceutical ingrdient in veterinary medicine to treat fleas and ticks in dogs. When patents expire between 2026 and 2066, it is expected that many products will be applied for approval as generic products, and reserch to establish accurate quality control methods must be conducted and managed. HPLC method was developed for the quantitative and qualitative of afoxolaner in veterinary medicinal products. The separation of active constituents for afoxolaner was achieved on a RP18 (4.6 x 150 mm, 5 μm) column using Water : Acetonitrile : MeOH (25:30:45 v/v/v) as mobile phase, with UV detection at 245 nm. The method was validated for specificity, linearity, accuracy and precision. All calibration curves showed good linearity (R2 of 0.999) within the concentration ranges (12.5 to 400 μg/mL). For accuracy, the recovery rate was calculated by spiking three concentrations of standard into the sample blank. The recovery rate was calculated to be 99.70~100.58%. Precision was measured 9 times repeatedly through intra-day, inter-day tests using standard. It showed excellent precision by satisfying the relative standard deviation of less than 2% both intra-day and inter-day. Limit of detection (LOD) and limit of quantitation (LOQ) were 2.0 μg/mL and 6.1 μg/mL, respectively. This method was successfully applied to analyzing afoxolaner drugs distributed in Korea. The HPLC method described in this study is accurate and reproducible and could be applied for the analysis of veterinary drugs of afoxolaner.