A simple, accurate reversed-phase high-performance liquid chromatography method was developed and validated for simultaneous determination of gliclazide (GLZ) and fluoroquinolone antibacterial levofloxacin (LVO). The method was developed by using a stainless steel analytical column , C18 (250,4.6 mm,5µm). The system was operated using a mobile phase consisting of methanol and phosphate buffer (pH 3.0) at a flow rate of 0.8mL minˉ¹ with ultraviolet detection monitored at wavelength 228 nm. The above method was validated using ICH analytical method validation guidelines. Utilizing HPLC techniques, an assay was intended to determine in vitro effects of levofloxacin on sulphonyl urea an anti-diabetic gliclazide. Obtained results were further verified with UV spectrophotometric method. Availability of gliclazide was reduced in the presence of levofloxacin. This in vitro analyses confirms the co-administration of gliclazide and levofloxacin and may serve for designing further in vivo studies.