Beer is the third most consumed beverage after coffee and tea, and therefore relies on strict quality assurance procedures to ensure consistent, high quality beer and consumer satisfaction. During brewing, yeast are exposed to a variety of stressors and must be able to respond to fluctuations in the environment such as pH, ethanol concentration and nutrient content. The re-use of yeast in subsequent fermentations (known as serial repitching) and their ability to maintain viability and vitality is also important for brewing performance. Recently, the applicability of molecular profiling using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in combination with Biotyper software was investigated for identification of beer spoilage microorganisms from routine biological quality control samples in a South Australian brewery. To expand the Biotyper platform and allow more comprehensive quality control practices, we assessed the monitoring of yeast fermentation performance using MALDI-TOF MS and incorporation a specific yeast health library into the database. Reference mass spectrum profiles of lager yeast (Saccharomyces pastorianus) exposed to osmotic, oxidative, ethanol, cold and nutrient stress were established and integrated into the Biotyper library. The applicability of monitoring yeast health was assessed by testing samples obtained from key points in production from a local South Australian brewery and the proteome analysed. Proteins that differentiate these stressed states from healthy yeast were subsequently identified using liquid chromatography tandem-mass spectrometry (LC-MS/MS). This technology will aid in improving brewing production, reducing economic waste and accelerate the growth of the industry.