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Fluoroquinolones antibiotics are more effective pathogens resistant to other antibacterial, thus being regularly prescribed drugs in human and veterinary medicine. Many fluoroquinolones posses one or two stereogenic centers giving rise to enantiomers whose pharmacological properties and toxicity are often different. At the successive stages of drug discovery, the enantiomers of any chiral molecule must be isolated and analyzed and their enantiomeric purity determined. The present review summarizes recent developments on chiral separation of fluoroquinolones using chromatographic and electrophoretic techniques.