<p>Sulfhydryl compounds are critical to the taste, flavor, and nutritional value of wine. However, reports on the detection and metabolism of thiol enantiomers in wine remain scarce. To address this gap, a novel UHPLC-HRMS method for the simultaneous detection and chiral resolution of three thiol enantiomers (DL-GSH, DL-Cys, DL-Hcy) in wine has been established, utilizing the chiral derivatization probe NCS-OTPP labeling. The method achieved complete separation of the three compounds within 25&#xa0;min (Rs = 1.70–1.83), with linearity (R² ≥ 0.9992), intraday precision ranging from 0.83% to 4.06%, and average recovery rates between 85.9% and 108%. Initial detection of thiol enantiomers in wines from ten different manufacturers revealed the presence of all three compounds, with their concentrations varying across different wine types. In red wine, L-Hcy was the most abundant, while D-Cys was the least. For white wine, L-GSH had the highest concentration, and D-Hcy had the lowest. Furthermore, principal component analysis (PCA) and VIP values of sulfhydryl compounds effectively differentiated red and white wines from various regions. To extend the applicability of the method and to gain insights into the in vivo behavior of wine-derived thiol compounds, the method was further applied to human urine as a biological matrix. Following wine consumption, changes in thiol levels were monitored, and metabolic fitting curves were constructed based on urinary data. The results showed that the content of thiol enantiomers in the body increased initially and then decreased after wine consumption. This study provides a robust scientific foundation for wine quality evaluation and offers valuable data for further investigation into the physiological effects of chiral sulfhydryl compounds in the human body.</p> Graphical abstract <p></p>