A number of polyfunctional thiols are well-recognised as some of the most potent aroma compounds in nature and have been identified in various foods, and beverages such as wine. These potent character impact compounds are present at nanogram-per-litre levels and are particularly challenging to measure. Where present, enantiomeric forms having different odour qualities further complicate the analysis. In this work, a chiral high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method using stable isotope dilution analysis (SIDA) has been developed and validated for the individual enantiomers of 3-sulfanylhexan-1-ol (3-SH, 1) and its O-acetate, 3-sulfanylhexyl acetate (3-SHA, 2), in wine after extraction as their 4-thiopyridine derivatives. Authentic derivatised thiols were first synthesised and used for chiral column screening and method development with polysaccharide-based HPLC phases. Validation of the method using a Lux Amylose-1 column for quantification of the enantiomers of 1 and 2 gave linear calibrations, and excellent figures for accuracy (recovery: 90%–111%, Z-score: −1.8–1.9), precision (RSD_r<8%), and limits of detection (0.7 ng L⁻¹ or less) in model media and different wine matrices. The newly developed method was applied to determine the enantiomer profiles of 1 and 2 in range of commercial wines.