There is growing interest in mapping exchange dynamics between Watson–Crick and Hoogsteen conformations across different DNA contexts. However, current methods are ill-suited for measurements at a large scale because they require isotopically enriched samples. We report that Hoogsteen dynamics can be measured on unlabeled samples using 1H CEST experiments, which have higher throughput and lower cost relative to conventional methods and also provide new insights into Hoogsteen dynamics.