Purpose: A sensitive, specific and efficient Ultra-Performance Liquid Chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the simultaneous determination of Quinolones by dried blood spots (DBS) is presented. Method: Human Red Blood Cell(RBC) were centrifuged and subsequent cells were washed with physiological buffer and added a precise volume of serum to produce blood with the hematocrit(Hct) 35%. Dried blood spots were punched out of a collection paper. The analytes were extracted by punching out disc and sonicated with a extraction buffer containing the Internal Standard. Separation was achieved on a Waters ACQUITY UPLC BEH C8 1.7μm Column using gradient condition. Running time was 10 min. For quantification, multiple reaction monitoring (MRM) chromatograms were acquired with MassLynxTM MS software (Waters). Mass transitions of m/z 362.18 → 261.03 for ofloxacin, m/z 402.21 → 109.92 for moxifloxacin and m/z 376.19 → 261.01 for gatifloxacin were optimized. Result: In this study, a UPLC-MS/MS system was used, which greatly improved the ion chromatography separation and peak shape. The method was linear (＞0.9992) from 0.2 to 10μg/mL with a linear equation. The intra-day precision (CV%) and accuracy (RE%) for 3 QC samples of Quinolones were ＜15% and ＜115%, respectively. Recovery of Quinolones by DBS was low (＞30%) but recovery coefficient variation(CV%) was ＜15%. Conclusion: The validated method was specific, sensitive, linear, accurate and precise and can be used to quantify Quinolones in human dried blood spots (DBS) for clinical studies.