Double-strand breaks are very potent inducers of DNA recombination. There is no recombination between DNA molecules unless one or two DNA strands are broken. It has become feasible to introduce double-strand breaks at specific chromosomal loci by using dedicated, redesigned endonucleases with altered DNA-binding specificities. Such breaks are mainly repaired by error-prone nonhomologous recombination pathways in somatic cells, thus frequently producing mutations at the preselected chromosomal sites. Although the art and science of reengineering protein properties have been advancing quickly, an empirical validation of new endonucleases in a particular experimental environment is essential for successful targeted mutagenesis experiments. This chapter presents methods that were developed for a comprehensive evaluation of the DNA-binding and DNA-cutting activities of homing endonucleases in maize cells; however, they can be adopted for similar evaluation studies of other endonucleases and other plant species that are amenable for Agrobacterium-mediated transformation.