The proportion of ingested anthocyanins to reach the systemic circulation is reported to be a small percentage of their ingested dose. This may be due to physiochemical degradation in vivo or following routine sample treatment. Therefore, this study aimed to quantitatively investigate the effect of anthocyanin structure on their stability under simulated (in vitro) physiological conditions and to assess their degradation and recovery following routine preanalytical sample extraction and storage. It was demonstrated that B-ring hydroxylation mediated the degradation of anthocyanins to their phenolic acid and aldehyde constituents, successful anthocyanin extraction is dependent on both sample preparation technique and anthocyanin structure, and anthocyanins are stable through multiple freeze-thaw cycles. These data indicate that significant portions of ingested anthocyanins are likely to degrade to phenolic acids and aldehyde in vivo. Consequently, these compounds should be the target of future bioavailability and bioactivity studies to establish the true occurrence and impact of anthocyanins on human health.