A simple analytical procedure, magnetic solid phase extraction combination to HPLC, has been developed for the analysis of gemifloxacin in human plasma and breast milk. Before chromatographic separation, magnetic solid phase extraction has been applied for sample preparation which is currently preffered extraction technique accordingly its simple, fast and efficient procedure. Fe3O4 magnetic nanoparticles have been used as magnetic adsorbents, the adsorption process has been optimized. RP C18 column has been used with mobile phase composed of acetonitrile-10 mM orthophosphoric acid including 1 mL/L triethylamine (60:40) by isocratic elution with flow rate of 1.3 mL/min. Calibration curve is linear over the range of 0.5-30 μg/mL and 0.5-20 μg/mL for plasma and breast milk, respectively. LOD and LOQ has been found to be 0.15 and 0.5μg/mL for both matrices. Intraday and interday RSD values are less than 3.57% for both assays.Moreover, the newly developed method provides fast, simple, cost reduced and sensitive assay for gemifloxacin.