Abstract: A method for simultaneous determination of residues of 40 glucocorticoids and 9 non-steroidal anti-inflammatory drugs (NSAIDs) in chicken tissues by HPLC-MS/MS was developed. Glucocorticoids and NSAIDs, which were widely used to treat animal diseases and improve growth rate, were ofen fed to animals illegally. Furthermore, this misuse had caused several risks to human health as toxic effects and hypertension. In order to increase food safety, maximum residue levels (MRLs) were established for glucocorticoids and NSAIDs in animal-derived food. The establishment of a high sensitivity and accurate method for glucocorticoids and NSAIDs detection is necessary. In this study, the target compounds in chicken breast sample were extracted by 90% acetonitrile aqueous solution (V/V, containing 0.1% formic acid) and purified by the Oasis PRIME HLB, a new reversed phase SPE with only one pass-through clean-up step. Comparing with traditional solid phase extraction, the new SPE eliminates pre-equilibration and conditioning steps maked a faster workflow. In addition, it had an excellent capacity for removing matrix interferences, particularly phospholipids. The chromatographic separation was performed on a Waters Acquity UPLC BEH column (2.1 mm×100 mm×1.7 μm) and gradient eluted by 0.1% formic acid and acetonitrile and analyzed by liquid chromatography tandem mass spectrometry in multiple reaction monitoring (MRM) mode via positive electrospray ionization．All the compounds were calibrated by the external method. To minimize the matrix effect, this experiment results were quantified by using the matrix-matched calibration curve. The results showed that the linear ranges of 49 drugs were 2-100 μg/L and the correlation coefficients were above 0.996. The recoveries were between 60.2% and 111.7% with the relative standard deviations from 1.3% to 12.0% (n=6) 。The limits of detection were 0.1-0.5 μg/kg, and the limits of quantification were 0.3-1.5 μg/kg, respectively. The method is easy to operate, sensitive, accurate, and is suitable for the simultaneous determination of glucocorticoids and NSAIDs residues in chicken tissues.