A new antiparasitic drug called afoxolaner targets the glutamate and gamma-aminobutyric acid receptors of acarine insects. The isoxazoline class of chemicals has been used as an active pharmaceutical ingredient in medications that veterinarians have prescribed to treat dogs for flea and tick infestations. There is a racemic combination of afoxolaner, which has a chiral center at the isoxazoline ring. To confirm that afoxolaner is a racemic mixture as shown by specific rotation and to ascertain the enantiomeric purity of single enantiomer samples, a normal phase chiral high performance liquid chromatography analytical method has been devised. The optimized procedure used a HypersilODSC18 column with a 150 *4.6 mm I.D of 5µ diameter kept at 35°C. Acetonitrile/MeOH (90/10, v/v) was used as the mobile phase for the gradient elution analysis of the samples. The UV calibration has shown 266 nm as detecting wavelength. The resolution and selectivity factors were 5.0 and 1.54, respectively and the two enantiomers were successfully separated in less than 10 minutes. The analytical method was properly validated for its intended usage in accordance with ICH criteria. A simple rapid and chiral phase RP HPLC technique was developed for enhanced separation of Afoxolaner which is sensitive and ecofriendly. Thus the method developed was found to be validated as per ICH guidelines.