Shiraz, also known as Syrah or Hermitage, is one of Australia's most popular red wine varieties both domestically and internationally. Black pepper aroma and flavor are important to some Australian Shiraz red wine styles. Recently, rotundone (a bicyclic sesquiterpene) was identified as the potent aroma compound responsible for pepper aromas in grapes, wine, herbs, and spices, including peppercorns. Here the development, optimization, and validation of the analytical method for the quantitative determination of rotundone in grapes and wine are described and discussed. The method is precise, accurate, robust, and sensitive with a subpart per trillion limit of quantitation. The method uses stable isotope dilution analysis with d(5)-rotundone as internal standard, solid-phase extraction and microextraction, and gas chromatography-mass spectrometry.