This master thesis deals with the development of method for free fatty acids determination in natural and processed cheese. In the theoretical part the possibilities of extraction, fractionation and determination of lipid fractions, characterization of fatty acids and various methods of their determination are described, as well as determination of free fatty acids. The main scope of the experimental section is optimization and validation of the selected method for determination of free fatty acids in various cheese matrices. For extraction of lipids from the sample the method with the mixture of diethylether and petrolether according to ČSN EN ISO 1735 was selected. Thin-layer chromatography and solid phase extraction were tested and compared for separation of free fatty acids. The method according to ČSN EN ISO 5509, using methanol solution of bortrifluoride, was applied for esterification. Fatty acid methyl esters were determined by gas chromatography with flame-ionization detection.