Breast cancer is the most common malignant disease in women of the developed world. The most common type of breast cancer is HR+/HER2-. The development of tumor resistance to older drugs has led to the need to develop new ones, such as cyclin-dependent kinase inhibitors. These drugs are always used in combination with aromatase inhibitors or fulvestrant. Bioanalytical methods for therapeutic drug monitoring enable precise and individual drug dosing, necessary due to differences in pharmacokinetics and a narrow therapeutic window of the drug. Following the ICHM10 guidelines, in this work a new bioanalytical method for detection of palbociclib and fulvestrant was validated, expressing linearity, accuracy, precision, recovery and matrix effect. Solid-phase extraction technique was used to isolate analytes from the plasma of healthy volunteers. The analysis was performed by liquid chromatography coupled to a mass spectrometer with a triple quadrupole analyzer. The method was validated in the concentration ranges of 30 - 300 ng/mL for palbociclib and 10 - 100 ng/mL for fulvestrant. Linearity (R > 0.9993), precision (RSD < 1.85) and accuracy (accuracy range from -10.42% to -3.92%) were demonstrated within the concentration range. Recovery for palbociclib is greater than 91.17%, while for fulvestrant it is greater than 77.52%. Matrix effect for PAL is 10.25% and 6.92%, at the lower and higher measured concentration, and for FUL it is -11.99% and -9.23%. Finally, the concentrations of palbociclib and fulvestrant in plasma samples of real patients were determined, which demonstrated the applicability of the method in therapeutic drug monitoring.