The aim of this study was to develop a rapid HPLC method with fluorescence detection for the separation of five fluoroquinolones in milk: norfloxacin (NFX), ofloxacin (OFL), moxfloxacin (MOX), ciprofloxacin (CFX), enrofloxacin (EFX) and the validation of a HPLC method for the determination of NFX and CFX in milk. The separation was performed on a Phenomenex Luna C 18 column, 150x4, 60 mm, 3 mm, having as mobile phase a mixture of: A = 20 mmol KH2PO4 tetra methyl ammonium hydroxide 10% brought to pH 3 with H3PO4 85% and B = methanol in linear gradient of concentration. Mobile phase flow rates were variable from 0.7 to 0.8 ml/min. For the determination of NFX and CFX flow rate was set at 0.8 ml/min. For determination, a fluorescence detector was used (excitation at 278 nm and emission at 455 nm). Analysis of milk samples was done after protein precipitation with 20% perchloric acid. In these conditions, the chromatographic retention times of the 5 fluoroquinolones were: MOX (rT = 4.31 min), EFX (rT = 6.14 min), OFL (rT = 8.05 min), CFX (rT = 9.24 min) and NFX (rT = 10.73 min). The method is linear and specific for the concentration range of 10-1000 ng/ml milk. The method developed shows good separation of the five fluoroquinolones and allows a rapid screening of their content in milk. A simple method was validated for the determination of ciprofloxacin and norfloxacin in milk, the analytical performance of the method was verified through the most important validation parameters.