Double-strand breaks (DSBs) disrupt DNA integrity and cause genomic instability and cancer, mutations, or cell death. Among the pathways utilized by cells of higher eukaryotes to repair this lesion, nonhomologous end-joining (NHEJ) is the most dominant. The biochemical characterization of NHEJ has significantly benefited from in vitro plasmid end-joining assays that can complement and extend information obtained from genetic studies. There is evidence that several factors involved in DNA-PK-dependent NHEJ remain to be identified. In addition, under certain circumstances, cells utilize backup pathways of NHEJ that depend on unknown factors to remove DSBs. Characterization of these putative factors will benefit from plasmid-based assays of DNA end-joining. Here, we describe a protocol for in vitro end-joining using plasmid DNA as substrate. The required procedures include: (1) preparation of HeLa-cell nuclear extract; (2) preparation of plasmid substrate DNA; (3) assembly of in vitro DNA repair reactions; and (4) product analysis by gel electrophoresis. The assay is powerful and easy to perform, but one should be aware that it represents an oversimplification, as it does not consider the in vivo organization of DNA into chromatin.