Imatinib mesylate is used to treat various cancerous diseases. Lately, investigations have focused on the enhancement of chemotherapeutic agents. Thus, rifampicin is a promising candidate due to its chemosensitizing potential beyond its anti-infectious effects. In this study, a reliable separation method for imatinib mesylate and rifampicin have been developed. The HPLC analysis were performed on a C18 column (150 x 4.6 mm, 3 μm particle size) at 25 °C. The best UV decetection was observed at 254 nm. The mobile phase was set as acetonitrile and TEA/phosphate buffer (pH: 7.04; 0.1 M) (50:50, v/v) with isocratic elution. The flow rate was set as 0.8 mL/min. The method validation was performed according to the international guidelines with respect to selectivity, linearity, precision and accuracy, recovery and sensitivity. The detection and quantification limit of the method were 0.63 μg/mL and 1.90 μg/mL, respectively for imatinib mesylate, and 3.04 μg/mL and 9.22 μg/mL for rifampicin. The method was linear in the range of 10–90 μg/mL with determination coefficients (r2≥0.99) for both drugs. Precision, accuracy and recovery values (RSD<3%) of the method was convincing. Considering the various usage of imatinib mesylate and rifampicin, the developed method is applicable to different dosage forms.