RNA tertiary structure and protein interactions can be detected by protection from hydroxyl radical cleavage. Generation of hydroxyl radicals with a synchrotron X-ray beam provides analysis on a short timescale (50 msec to 100 sec), which enables the structures of folding intermediates or other transient conformational states to be determined. This unit provides detailed instructions on the use of the synchrotron beamline for hydroxyl radical footprinting.