A robust quantification method for polyfunctional thiols in wine is highly desirable due to their aromatic importance to many varietal wines. However, analytical determination of these compounds at levels near or below their ng/L sensory thresholds is difficult due to th e complexity of a wine matrix, and poor detectability, poor chromatographic behavior, and high reactivity of the thiol functional group. An improved method for the quantification of thiol contributors to wine aroma by headspace solid-phase microextraction (SPME) coupled to gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) has been developed. Thiols are converted to their corresponding pentafluorobenzyl derivatives by extractive alkylation, the organic layer dried under nitrogen, and reconstituted in aqueous buffer prior to SPME analysis. Optimal extractive alkylation parameters (pH 12) and SPME parameters (70 °C, 60 minutes) were determined by response surface area modeling. Using 40 mL wine samples, achievable limits of detection for 4-methyl-4-mercapto-2-pentanone (4-MMP), 3-mercaptohexanol (3-MH), and 3-mercaptohexyl acetate (3-MHA) were 0.9 ng/L, 1 ng/L, and 17 ng/L, respectively. Standard addition of these thiols in a model wine system showed good linearity (R2 > 0.99 for all thiols) over two orders of magnitude and applicability of method to a commercial wine matrix was confirmed through recovery (90-109%) and precision (5.4% < RSD < 11.1%) experiments. The method is scalable by pooling organic extracts over a sample size range of 10 to 160 mL, resulting in corresponding improvements in limits of detection. The method was validated using Riesling, Gewürztraminer, Cayuga White, Niagara, Rosé, and Sauvignon blanc wines from the Finger Lakes wine region in upstate New York. This method also permits measurement of low-molecular weight thiols responsible for certain aromatic defects in wine, such as H2S and CH3SH, in the same analysis.