The present study is interested to develop a simple, rapid and accurate spectrophotometric method for determination of sodium flucloxacillin (fluc) in pure form and pharmaceutical formulations. The charge-transfer (CT) interactions between sodium flucloxacillin as electron donor and chloranilic acid (CLA), dichloroquinone 4-chloroimide (DCQ), 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and 7,7,8,8 tetracyano-p-quinodimethane (TCNQ), as π-electron acceptors have been investigated spectrophotometrically. Different variables affecting the reaction were studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9979–0.9995) were found between the absorbance and the concentration of the drug in the range 16–880μgml⁻¹. The proposed methods were applied successfully to the determination of the examined drug either in pure or pharmaceutical dosage forms with good accuracy and precision. The formation of the CT-complexes and the sites of interaction were confirmed by elemental analysis CHN, UV–vis, IR, ¹H NMR and mass spectra techniques. Based on Job's method of continuous variation plots, the obtained results indicate the formation of 1:1 charge-transfer complexes with the general formula [(fluc)(acceptor)]. Statistical analysis of the obtained results showed no significant difference between the proposed method and official method.