Práce bylo zaměřena na vyvinutí vhodného postupu úpravy vzorků krevního séra, tak aby v nich bylo možné metodou HPLC/MS stanovit různá antidiabetika. V práci jsou posány různé metody úprav biologických vzorků, příklady analýz biologických vzorků pomocí HPLC/MS a krátce jsou zde rovněž popsány skupiny antidiabetik. Výsledkem práce jsou dvě metody přípravy vzorků a chromatografická metoda určená k jejich analyzování.The aim of this diploma thesis was development of a suitable procedure for determination of antidiabetic drugs by HPLC/MS method in blood serum sample. Theoretical part of this thesis describes common procedures for the pretreatment of biological samples by simple protein precipitation, liquid-liquid extraction and very popular solid phase extraction method. A substantial part of this chapter deals with the solid phase extraction ant its modifications. Diabetes and its complications is described in this chapter, too. Type 2 diabetes is treated by peroral antidiabetic drugs which are also mentioned here as well as a few examples describing biological sample preparation for subsequent determination of these drugs. Experimental chapter is devoted to the instrument and experimental settings, chromatographic separation parameters and mass spectrometry parameters. Sample procedures are described there. The Results and discussion chapter contains more detailed description of the development of suitable chromatographic separation conditions and also contains chromatogram samples. Ionization and fragmentation conditions are listed here, including fragmentation spectra of all used antidiabetics. Results of two methods of sample pretreatment, methods validation and model sample analysis results are presented here.