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Background: X22 is one of the imidazopyridine derivatives designed and synthesized in our laboratory. This compound showed excellent anti-inflammatory activity in LPS-stimulated macrophages. We previously investigated the biological activities of X22, including treatment of obesityrelated complications and retinal ischemia/reperfusion injury. Methods: In the present study, a reliable, rapid, and simple UPLC method was first established and validated for quantitative analysis of X22 in rat plasma. Plasma samples were prepared by protein precipitation and separated on an ACQUITY HSS T3 column with a gradient mobile phase consisting of acetonitrile and water containing 0.1% TFA at a flow rate of 0.3 mL/min. Results: Good linearity (R2>0.997) was achieved using weighted (1/x2) least-squares linear regression over a concentration range of 10 ng/mL to 1000 ng/mL with a lower limit of quantification of 10 ng/mL for X22. The average extraction recoveries were > 91.3 % for X22 and > 92.4 % for neohesperidin dihydrochalcone. The intra- and inter-precision values of the assay were both < 4.0 %. Conclusion: This method was successfully applied to a pharmacokinetic study on rats after administration of single intravenous and oral doses of 20 and 80 mg/kg, respectively.