Imatinib mesylate is a small molecule used in cancer therapy as a thyrosine kinase inhibitor. Dexketoprofen trometamol is a non-steroidal anti-inflammatory drug that has seen use in cancer therapy in combination with an anticancer drug to minimize tumor size and to reduce pain in patients. In the present study, imatinib mesylate and dexketoprofen trometamol were selected as potential model drugs to be used in combination. A new, simple and selective Ultra Performance Liquid Chromatography method was developed and validated to determine the drug substances in distilled water, in a pH 7.4 phosphate buffer and in Dulbecco's Modified Eagle Medium. The proposed method was developed using a BEH C-18 column with isocratic elution. A mixture of methanol:acetonitrile (80:20, v/v) and pH 9.5, 0.05 M ammonium acetate were (70:30, v/v) used as a mobile phase. Detection was carried out with a flow rate of 0.3 mL/min, a column temperature of 30 degrees C and an injection volume of 20 mu L. The method was validated considering linearity, accuracy, precision, specificity, robustness, detection limit and quantitation limit values, and was found to be linear in a range from 0.05 to 20.0 mu g/mL for the three different media.