A derivative UV-spectrophotometric analytical procedure was developed for determination of three 4-quinolone antibacterials: norfloxacin (NFX), ciprofloxacin (CFX), and sparfloxacin (SFX). The method depends on the complexation of Cu(II) with the studied compounds in aqueous medium. A third order, measurement was applied for their quantification. A linear correlation was established between the amplitude of the peak and concentration for all the studied drugs in the range of 15-80, 35-120, and 200-700 ng/mL, with minimum detectability (S/N = 2) of 1.0, 1.3, and 5.1 ng/mL for NFX, CFX, and SFX, respectively. The method was successfully applied for accurate, sensitive, and selective determination of the studied drugs in bulk and tablets formulation with average percentage recoveries of 99.22 +/- 0.55 to 100.33 +/- 1.60. The results obtained were favorably compared with those of the reference method. The method was also used to determine sparfloxacin in spiked human plasma and urine. The results obtained were satisfactory, accurate, and precise.