Varietal thiols are important contributors to the aroma profile of a wide range of beverages including beer. A methodology for the simultaneous extraction and subsequent analysis of the varietal thiols 3-sulfanylhexan-1-ol (3SH), 3-sulfanylhexylacetate (3SHA) and 4-methyl-4-sulfanylpentan-2-one (4MSP), and the non-volatile precursors 3-S-cysteinylhexan-1-ol (Cys-3SH) and 3-S-glutathionylhexan-1-ol (GSH-3SH) in beer was developed and validated for the first time. The method, which utilises LC-MS/MS, was successfully tested on nine commercial beers, showing it to be widely applicable to this matrix. The syntheses of the novel internal standards d8-3SH and d8-3SHA, which allow a robust and accurate quantification of the hydronated congeners, are also reported. The simplicity of the QuEChERS-based (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction allows for rapid, high throughput analyses suitable for both research and industry applications.