Polyphenols in pigmented cereals are believed to enhance health outcomes through their antioxidant properties. This study aimed to characterise polyphenols from Hordeum vulgare (purple barley), Triticum turgidum (purple wheat) and Triticum aestivum (blue wheat) in order to evaluate their bioaccessibility and antioxidant activity. An ultra-high performance liquid chromatography mass spectrometry coupled with an online 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) system was used to identify the polyphenols and quantify their relative antioxidant levels. Simulated gastrointestinal digestion of the cereals allowed for the assessment of polyphenol bioaccessibility using benchtop assays. Between cereals, the bioaccessible phenolic content was similar following digestion, but the antioxidant activity was significantly different (purple barley > purple wheat > blue wheat; p < 0.01). Among the polyphenols identified, flavan-3-ols and anthocyanins were the least bioaccessible whereas flavones were the most bioaccessible after digestion. This study demonstrated that these pigmented cereal varieties are sources of bioaccessible polyphenols with antioxidant activity. These findings may aid in utilising these pigmented grains for the future design and development of novel functional food products with enhanced health properties.