Objectives: The aim of this study was to develop four simple and accurate spectrophotometric methods for determination of flucloxacillin-sodium in binary mixture with ampicillin-trihydrate without separation. Methods: One of them was a univariate constant center method and the other three methods were multivariate chemometric methods named; Savitsky-Golay filters, continuous wavelet transform of ratio spectra and wavelet transform of first derivative of the ratio spectra. Results: The proposed methods adopted for selective determination of flucloxacillin-sodium and obey Beer's law in the range (2-20 µg mL^-1). Conclusion: The proposed methods were simple, rapid, economic, accurate and precise; they were successfully applied for the determination of flucloxacillin-sodium in pure form and in pharmaceutical preparations.