Anastrozole and ribociclib are used in combination as initial endocrine therapy for ER-positive breast tumors in postmenopausal women. Anastrozole works to reduce the available estrogen in the body, which is necessary for tumor proliferation, while ribociclib slows down the cell cycle of tumor cells, thereby reducing the possibility of resistance to therapy. The objective of this work was to develop a selective analytical method for the simultaneous analysis of ribociclib and anastrozole in human plasma as a basis for further development of a sensitive method for determining these drugsin patients' plasma. XBridge (Waters) Phenyl (4.6 x 150 mm, 3.5 μm) stationary phase was used in high-performance liquid chromatography. The column temperature was set at 25 °C. Various mobile phase combinations of acetonitrile and water with 0.1% formic acid were tested, including isocratic elution, gradient washing, and different mobile phase flow rates. The most favorable method was gradient elution, where the ratio of water to acetonitrile changed from 75:25 to 15:85 (V/V) during the first 6 minutes of the analysis, with a flow rate of 0.5 mL/min. The retention times for ribociclib and anastrozole were 6.4 min and 8.7 min, respectively. A diode array detector was employed for detection. The UV spectra of both analytes and the selected detection wavelengths of 220 nm for anastrozole and 270 nm for ribociclib were determined. To mitigate the solvent effect when using acetonitrile, a mixture of methanol and water in a ratio of 50:50 (V/V) was utilized as the optimal solvent for the analysis. Phenazone was chosen as an internal standard due to its similar behavior to the analytes when processing complex biological samples, its convenient retention time (6.16 min), and properties (polarity, molecular weight) close to those of the analytes. The plasma is extracted from whole blood by centrifugation using EDTA as an anticoagulant in accordance with the standard procedure. Various methods involving different ratios of methanol and acetonitrile, with and without the addition of phosphoric acid, were tested for matrix removal, which refers to the deposition of plasma proteins. The addition of phosphoric acid to organic solvents did not significantly reduce interferences, while protein precipitation with methanol required a large volume of solvent. The protein precipitation method with acetonitrile in a volume ratio of acetonitrile:plasma of 4:1, followed by solvent evaporation to dryness and resuspension in 50% methanol, proved to be optimal. In accordance with the ICH guidelines for the validation of bioanalytical methods, the selectivity of the developed method was tested using six blank plasma samples from six different donors.