The aim of this work is to select the optimal pre-analytical processing of the plasma samples and the development of a novel method for the simultaneous quantification of anastrozole (ANA) and palbociclib (PAL) by high performance liquid chromatography coupled to a mass spectrometer (LC-MS). The methods of protein precipitation, solid phase extraction (SPE), phospholipid removal and liquid-liquid dispersive microextraction (DLLME) were compared. In order to determine the optimal conditions for solid phase extraction, the values of extraction efficiency and matrix influence of six different sorbents in combination with different eluents were compared. The best results were observed during extraction on a column with C18 sorbent, the eluent in the extraction was acetonitril (ACN), then 5% (v/v) ammonia in methanol. In order to determine the optimal conditions of DLLME, the values of extraction efficiency and matrix influence of five DLLME variants were compared. The best results were observed with the DLLME of the water sample, where the extraction agent is clorophorm, the dispersion agent is isopropanol, and the ionic strength of the solvent is increased by the addition of citrate salt. The samples have been analyzed and quantified by a novel LC-MS method. Reverse phase chromatography was performed on a biphenyl column with binary gradient elution in which the mobile phase consists of 0.1% formic acid disolved in water and 0.1% formic acid disolved in ACN. MS analysis was performed with electrospray ionization in positive imaging mode by monitoring of ion transitions m/z 448,2→380,0 and m/z 448,2→319,5 for PAL, and m/z 294,1→225,1 and m/z 294,1→210,0 for ANA. Analytes were identified based on their mass spectra and retention times. Quantitative analysis was carried out by integrating the chromatogram spikes.