AbstractSelf-cleaving ribozymes are catalytic RNAs that cleave their own sugar phosphate backbone site-specifically. While most ribozyme classes were discovered serendipitously, only four classes were found through targeted methods. We developed an RNA-seq-based strategy, called cyPhyRNA-seq, to capture both ribozyme cleavage fragments in a targeted fashion. This method can be used to study ribozyme activity on a global scale and has the potential to uncover new self-cleaving ribozyme classes.