Isothiocyanates are degradation products of glucosinolate hydrolysis. Glucosinolates are phytochemicals found mostly in plants from the cabbage family. Isothiocyanates are potentially biologically active, which is why methods for their determination are being developed. In this thesis, a method for the determination of 2-methoxyphenyl isothiocyanate, 3-methoxyphenyl isothiocyanate and 4-methoxyphenyl isothiocyanate was developed. Gas chromatography-tandem mass spectrometry (GC-MS/MS) was applied for the separation of selected isothiocyanates and qualitative and quantitative determination of separated compounds. Standards of 2-, 3- and 4-methoxyphenyl isothiocyanate and solvent n-hexane were used for the preparation of solutions. Gas chromatography was optimized by changing the oven temperature program rate (20 °C min -1 ). Triple quadrupole mass spectrometer, in MRM scan mode, was used for monitoring specific fragmentation reactions (quantifier and two qualifiers for each compound). Mass spectrometry was optimized by determination of the collision energy with nitrogen for each specific reaction of fragmentation. Different approaches of preparing the calibration curve working solutions were examined (forward pipetting with mechanical pipettes was selected) as well as different concentration ranges (range 2,5 μg mL -1 – 20,0 μg mL -1 was selected) were examined for making calibration curves. The validation parameters tested: accuracy, precision, specificity, linearity, the limit of detection, the limit of quantification and stability, were proven satisfactory. The method was successfully applied in the analysis of a synthetic sample of known concentration. This ensures the suitability of the method for the individual determination of 2-, 3- and 4-methoxyphenyl isothiocyanates in the mixture.