Ribociclib is an antineoplastic indicated for treating postmenopausal women with locally advanced or metastatic breast cancer that is HR-positive and HER2-negative. It is used in combination with hormone therapy that reduces the effect of estrogen, one such drug being letrozole (an aromatase inhibitor). During the development of bioanalytical methods for therapeutic monitoring of these drugs, the use of internal standards is crucial to eliminate errors arising during sample preparation for analysis. The optimal internal standard for this purpose is an isotopically labeled version of the molecule to be quantified, with deuterium being preferred due to the abundance of hydrogen in organic molecules.The main aim of this research was to optimize fragmentation conditions for deuterated letrozole and deuterated ribociclib using a triple quadrupole mass spectrometer. Optimization involved determining the optimal fragmentor voltage, selecting the fragments with the most intense signal, and determining the optimal collision energies to generate these fragments. Positive electrospray ionization of deuterated letrozole forms a molecular ion with m/z 290.1. An optimal fragmentor voltage of 80 V gives the highest signal intensity for this molecular ion. Fragments with m/z values of 194.2 and 221.0 are most suitable for analysis, with optimal collision energies of 40 eV and 10 eV. Deuterated ribociclib in the ion source gives a molecular ion with m/z 441.2, with an optimal fragmentor voltage of 160 V. Optimal fragments with m/z 322.1, 252.2, 294.1, and 373.1 have the highest signal intensity at collision energies of 35 eV, 60 eV, 45 eV, and 20 eV. The research also showed that positive electrospray ionization is more effective for analyzing these deuterated compounds compared to negative electrospray ionization, as it generates fragments with more intense signals.