Fluoroquinolones (FQs) are among the most important antibacterial agents (synthetic antibiotics) used in human and veterinary medicine. They are employed against all bacterial infections, particularly against urinary tract infections and acute respiratory diseases. In the last decade, there was no review covering all different analytical methods used for the determination of fluoroquinolone antibiotics. The present review presented recently published different electrophoretic and chromatographic methods for determination of seven fluoroquinolones (ciprofloxacin, gatifloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, and sparfloxacin). Presented applications concern analysis of chosen fluoroquinolones in pure forms, different pharmaceutical formulations, biological fluids, and environmental samples.