A new, sensitive, and selective high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of moxifloxacin (MOX) in human breast milk. MOX was precolumn derivatized with fluorescamine; the fluorescent derivative was separated on an RP C18 column using a mobile phase composed of acetonitrile-10 mM orthophosphoric acid by isocratic elution with flow rate of 0.5 mL min(-1). The method was based on the measurement of the derivative using fluorescence detection at 481 nm with excitation at 351 nm. The calibration curve was linear over the range of 1-40 mu g mL(-1). Limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.3 and 1 mu g mL(-1), respectively. Intra-day and interday repeatabilities were less than 3.15%.