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A novel methodology has been developed for simultaneous separation of ciprofloxacin (CPFLX), gatifloxacin (GTFLX), levofloxacin (LVFLX), moxifloxacin (MFLX) and sparfloxacin (SPFLX) fluoroquinolone antibiotics (FQs), using capillary zone electrophoresis (CZE) with UV detection at 282
nm. Electrolyte composition was optimized through the variation of the Tris/hydrochloride and sodium tetraborate buffer mixture. The electrolyte consisted of a 25
mmol
L
−1 Tris/hydrochloride and 15
mmol
L
−1 sodium tetraborate buffer mixture resulting in pH 8.87. All analytes were separated in less than 3
min. The proposed method was applied to the separation of FQs in pharmaceutical formulations, and the assay results were within 95–105% of the label claim.