The Thermo Scientific Dionex UltiMate 3000 LC system is applied for assay and related substances analysis of Flucloxacillin. The analysis was performed on a Thermo Scientific Hypersil GOLD aQ HPLC column using the method described in the EP 8.0 and KP X. The results obtained for resolution and % RSD met the criteria stated in the EP and KP. The results obtained for resolution between the peaks due to Cloxacillin and Flucloxacillin was 5.9 (EP and KP criteria of minimum 2.5) and %RSD for flucloxacillin (0.1 mg/mL) was 0.1 (EP & KP criteria of maximum 1).