Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA–protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA–protein complexes; this technique can provide structural detail that is not achievable using DNase I footprinting. Hydroxyl radical footprinting experiments can be carried out using readily available and inexpensive reagents and lab equipment. This method involves using the hydroxyl radical to cleave a nucleic acid molecule that is bound to a protein, followed by separating the cleavage products on a denaturing electrophoresis gel to identify the protein-binding sites on the nucleic acid molecule. We describe a protocol for hydroxyl radical footprinting of DNA–protein complexes, along with a troubleshooting guide, that allows researchers to obtain efficient cleavage of DNA in the presence and absence of proteins. This protocol can be completed in 2 d.