Abstract: OBJECTIVE To establish a method based on two-dimensional online cleanup mode with three columns coupled to liquid chromatography-tandem mass spectrometry for the determination of eight quinolones residues(norfloxacin, enoxacin, ciprofloxacin, lomefloxacin, ofloxacin, mapofloxacin, shafofloxacin and obifloxacin) in chicken.METHODS Used the principle of size exclusion chromatography to make the matrix interfering substances directly being eluted without reservation.The target compounds of small molecules were retained for longer time on the chromatographic column.So the separation of target compounds and matrix was realized in this method.After excluding the interference of matrix, the target compounds were retained on the enriched column by the switching the valve.After the column collection, the two-dimensional chromatography wash switched to the initial state, and the target substances were separated and analyzed on the second two-dimensional column.After extracted with acetonitrile and an automated sample cleanup procedure with aqueous gel column, the eight quinolones residues in chicken samples were precisely determined in multiple reaction monitoring(MRM) mode by two-dimensional liquid chromatography-tandem mass spectrometry(LC-MS/MS).RESULTS The limit of quantification for the eight quinolones was 0.2 μg·kg^–1, the method produced linear results for eight guinolones from 4.0 to 100.0 ng∙mL^–1 with a linearity >0.991.The recoveries was in the range of 92.5%-109.5% with the relative standard deviations between 1.1% and 9.2%.Ofloxacin was detected in two batches of chicken among 67 batches, which were consistent with the results of the national standard test method, and the deviations were <5%.CONCLUSION This method is simple, rapid, accurate, and suitable for the simultaneous determination of the eight quinolones in chicken.