Abstract: Researchers describe the development of a Novel Method that is sensitive and reliable for detecting isoxazolyl penicillins (Cloxacillin (CLO) and Dicloxacillin (DCL)) in commercially available cow milk using High-Performance Liquid Chromatography with UV-Diode Array Detection (HPLC-DAD). CLO and DCL were extracted via deproteinization of milk samples, adjusting the pH to 4.5-5 using hydrochloric acid and then adding acetone/chloroform, followed by centrifugation and cleanup using a 0.45 μm nylon filter. CLO and DCL were separated using an Inertsil ODS-C₁₈ column (4.6x250 mmx3.5 μm) via elution with a mobile phase consisting of 0.05 M phosphate buffer solution at pH 4.7 and acetonitrile at 22°C. Recovery of CLO and DCL was 96.47-106.94 and 97.76-104.86%, respectively. The limit of quantifications were 23.15 μg kg^-1 for CLO and 2.28 μg kg^-1 for DCL which are lower than the limits for residues in cow milk established by the European Union, indicating that the method is suitable for performing routine analysis. In order to determine this Novel Method’s utility in a laboratory environment for analyzing real raw milk samples 100 raw milk samples were obtained from raw milk tanks belonging to milk producers. CLO and DCL were not detected in any of the samples. The present findings indicate that the Novel Detection Method is simple, rapid, sensitive and reliable, as compared to other detection methods and that as it uses fewer chemicals and does not require derivatization it could be considered the preferred method.