This paper reports the multi-residue determination of quinolones included in the European Union regulations (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin and flumequine) in bovine milk, using liquid chromatography with ultraviolet (LC-UV), fluorescence (LC-FD), mass spectrometry (LC-MS) and tandem mass spectrometry detection (LC-MS/MS). The methods were validated, in line with EU requirements (Commission Decision 2002/657/EC). Linearity, decision limit (CCalpha), detection capability (CCbeta), detection limit (LOD), quantification limits (LOQ), recoveries, precision, selectivity and stability were determined and adequate results were obtained. Recoveries higher than 80% were obtained for all quinolones. The methods developed were used to quantify enrofloxacin and its main metabolite, ciprofloxacin, in milk from animals treated with enrofloxacin.