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 (CCα), detection capability (CCβ), 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.