Beer spoilage microorganisms present a major risk for the brewing industry and can lead to cost intensive recall of contaminated products, damage to brand reputation or in extreme cases, to the loss of business. Current microorganism detection procedures for bacterial and wild yeast contaminations involve cultivation-based enrichment and optical analyses, coupled with more recent molecular methods such as PCR, riboprinting, rRNA hybridization and antibody-based techniques. However, optical read-out methods are prone to misidentifications, whilst molecular methods like PCR are cost intensive. We investigated the applicability of molecular profiling using mass spectrometry in combination with the Biotyper software for the identification of beer spoilage microorganisms as a rapid and cost effective method for brewery quality control testing. Reference mass profiles for the five most common beer spoilage microorganisms (Lactobacillus brevis, Lactobacillus lindneri, Pediococcus damnosus, Pectinatus frisingensis and Megasphaera cerevisiae), four commercially-available brewing yeasts (top- and bottom-fermenting) and Brettanomyces bruxellensis wild yeast were established and incorporated into the Biotyper reference library. The method was validated by inoculation of these microorganisms into beer samples and identification using Biotyper. All tested bacteria, bottom- and top-fermenting and the wild yeast strain could be accurately identified and distinguished from one another, as well as from the other approx. 5,600 microorganisms present in the Biotyper database. The applicability of mass spectrometry profiling for the brewing industry was assessed by testing routine quality control samples from a local brewery, where contaminating microorganisms could be reliably identified. This renders the Biotyper platform a promising candidate for routine biological quality control within the brewing industry, especially considering the time-to-result is faster and analysis cost per sample is smaller relative to other molecular methods such as PCR.