Barley beers may be rendered gluten-free by specialized brewing processes. One controversial issue is whether these beers contain residual gluten peptides carrying at least one celiac disease (CeD)-active epitope, because routine methods to assess whether the beer is gluten-free like competitive enzyme-linked immunosorbent assays (ELISA) may fail to detect potentially harmful peptides. We used untargeted nano-liquid chromatography-tandem mass spectrometry (nanoLC MS/MS) to identify CeD-active peptides in 21 gluten-free barley beers and four regular or carbohydrate-reduced beers. While the G12 ELISA confirmed gluten levels below 20 mg/kg in all gluten-free beers, the R5 ELISA detected gluten levels in four beers exceeding this threshold, highlighting method discrepancies. nanoLC-MS/MS identified 44 CeD active peptides in seven gluten-free and four non-gluten-free beers, of which 17 CeD-active peptides had no ELISA epitope and therefore escape detection. Accurate quantitation of these peptides combined with in vivo toxicity assessment will be needed to provide guidance on clinical relevance.