Objectives: An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) assay has been developed and validated for the quantification of oxycodone in rat serum and brain tissue homogenates. Methods: A simple extraction method using methyl-t-butyl ether was used for sample extraction with oxycodone–D₃ as an internal standard (IS). A Symmetry^® C₁₈ column (100 mm x 4.6 mm, 5 µm) connected to a Phenomenex Luna^® guard column (4 x 3 mm, 5 µm) was used for chromatographic separation. The mobile phase was composed of a mixture of acetonitrile and 0.1 % formic acid in water (pH = 2.7) (15:85, v/v). The flow rate was 0.4 mL/min, the total run time was 5 min, and the injection volume was 10 µL. The column and autosampler temperatures were maintained at 60 °C and 25 °C, respectively. Results: The calibration curve for oxycodone was linear over the range of 10–2000 ng/mL. Oxycodone extraction recovery from rat serum and brain samples ranged from 90.78–105.85 % and 98.42–104.38 % with relative standard deviation (RSD) values of 0.94–3.87 % and 1.04–2.82 %, respectively. The inter-day accuracy values ranged from 87.67–104.83 %, while the intra-day accuracy values ranged from 86.95–105.67 %. Conclusion: This method can be used for the quantification of oxycodone in samples obtained from preclinical animal studies and has great promise for applications in the quantification of oxycodone in human biological matrices.