DNA double-strand break (DSB) repair in mammalian cells has been demonstrated to be complex, involving both homologous and nonhomologous processes. Although manipulation of chromosomal DSBs and analysis of their repair are possible (1 ; see Chapters 37 –39 ), this is usually time-consuming, requiring the establishment and expansion of cell lines. To circumvent this we have refined an extrachromosomal assay to study both homologous and non-homologous DSB repair processes in mammalian cells (2 ,3 ). The assay is not only useful to study general mechanisms of DSB repair in different mammalian culture systems, but can also be applied to the classification and characterization of molecular defects in repair-deficient mammalian cells (3 ).