Objective A comprehensive analytical method based on ultra performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS) with positive mode has been developed for the simultaneous determination of seven tetracyclines and fourteen quinolones in milk. Methods Milk samples were extracted by EDTA-McIlvaine (pH=4.0) buffer solution and then purified and enriched by Oasis HLB solid phase extraction cartridges. The separation was performed on a Waters ACQUITY UPLC^TM BEH C18 column (100 mm×2.1 mm i.d., 1.7μm) with gradient elution using methanol-acetontrile and water (containing 0.2% formic acid) at a flow rate of 0.2 ml/min. Results The average recoveries of this method for fortified samples were ranged from 89.2% to 119.1%, with relative standard deviations between 1.8% to 19.0%. The limits of quantification (LOQ) were ranged from 0.1 to 0.5 μg/kg. Conclusion Due to its high sensitivity and reproducibility, the method is suitable for the determination of seven tetracyclines and fourteen quinolones in milk samples.