Odoriferous compounds: 3-sulfanylhexanol, 3-sulfanylhexyl acetate, and 4-methyl-4-sulfanylpentan-2-one formed during the fermentation of precursors present in grape and must are the most important representatives of the group of varietal thiols. These compounds have a crucial role in the formation of aromatic profile of certain wines and a very low odor detection threshold. Due to the high reactivity, polarity, higher boiling points, and very low abundance in wine, isolation of varietal thiols represents tedious work, difficult to achieve by routine methods. Extraction of varietal thiols almost always involves sophisticated steps of specific extraction, derivatization, purification, and preconcentration. A series of procedures for derivatization of varietal thiols in Sauvignon blanc wine were conducted and validated. Derivatives of varietal thiols were isolated by solid-phase extraction and microextraction. Calibration was performed by standard addition approach combined with stable isotope dilution analysis. Derivatization and extraction of individual varietal thiols from model wine, neutral wines, and varietal wines was optimized. The developed method was successfully used for quantification of varietal thiols by gas chromatography – mass spectrometry with triple quadrupole. Quantification was preceded by the development of a multiple reaction monitoring method. For the first time, concentration of varietal thiols in the samples of Croatian Sauvignon blanc wine was determined. All the wines show thiol presence above the respective sensory thresholds. The influence of various antioxidants added during the vinification step on the aroma profile of wine and the concentration of varietal thiols was investigated.