Breast cancer is the most common malignant disease in women. Nowadays, its mortality rate is decreasing due to new therapeutic methods, despite a continuous increase in incidence. To ensure proper dosing of therapy that will maximize drug efficacy and minimize side effects, it is important to monitor drug concentrations in plasma. When analyzing biological samples, it is essential for the method to be sensitive enough to detect analytes at low concentrations and effectively distinguish them from interfering substances that are also present. In this study, the analytes were six breast cancer drugs: ribociclib, abemaciclib, and palbociclib as CDK4/6 inhibitors, anastrozole and letrozole as non-steroidal aromatase inhibitors and fulvestrant as a competitive ER antagonist. The use of MEKCMS/MS in the analysis of these drugs has not yet been described in the literature. The aim of this study was to determine the optimal MS/MS conditions to ensure satisfactory method sensibility and quantification of these drugs. The optimized conditions were: temperature (75-250 °C) and sheath gas flow (3-11 L/min), flow rate (2-12 μL/min) and sheath liquid composition - suitable organic solvent (methanol, isopropanol, ethanol, acetonitrile), its proportion (50-80%, v/v) and formic acid content (0-0.5%, v/v). Nozzle voltage (0-2000 V) and capillary voltage (2000-4000 V) were also optimized. The ESI source gas parameters which were optimized are temperature (200-350 °C), flow (5-11 L/min) and nebulizing gas pressure (6-14 psi). The following results were obtained: the optimal sheath gas temperature is 250°C, with a flow rate of 5 L/min. Methanol at a concentration of 70% proved to be the best organic solvent in the composition of the sheath liquid. The optimal formic acid content in the sheath liquid is 0.2%, with a sheath liquid flow rate of 1 mL/min. The optimal nozzle voltage is 2000 V, while the capillary voltage is 2500 V. The ESI source gas parameters are: temperature 350°C, flow rate 9 L/min, and nebulizing gas pressure 12 psi.