Beer spoilage microorganisms present a major risk for the brewing industry and can lead to cost intensive recall of contaminated products and damage to brand reputation. 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 the identification of beer spoilage microorganisms from routine brewery quality control samples. Reference mass spectrum profiles for top- and bottom-fermenting brewing yeast strains, wild spoilage yeast and common bacterial spoilage organisms were established, incorporated into the Biotyper reference library and validated by successful identification after inoculation into beer. The applicability and integration of mass spectrometry profiling using the Biotyper platform into existing brewery quality assurance practices within industry was assessed by analysing routine microbiology control samples from Coopers Brewery Ltd., where contaminating microorganisms could be reliably identified and distinguished from over 5,600 microorganisms present in the database. This renders the Biotyper platform a promising candidate for biological quality control testing within the brewing industry as a more rapid, high-throughput and cost-effective technology that can be tailored for the detection of brewery-specific spoilage organisms from the local environment.