DNA damage repair is essential for the maintenance of genetic integrity in all organisms. Unrepaired or imprecisely repaired DNA can lead to mutagenesis, cell death, or malignant transformation. DNA damage in the form of double-strand breaks (DSBs) can occur as a result of both exogenous insults, such as ionizing radiation and drug therapies, and normal metabolic processes including V(D)J recombination. Mammalian cells have multiple pathways for repairing DSBs, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). This chapter describes the use of reporter substrates for assaying the contributions of these pathways to DSB repair in mammalian cells, in particular murine embryonic stem cells. The individual contributions of NHEJ, HR, and SSA can be quantified using fluorescence and PCR-based assays after the precise introduction of DSBs either by the I-SceI endonuclease or by the RAG recombinase. These reporters can be used to assess the effects of genetic background, dominant-negative constructs, or physiological conditions on DSB repair in a wide variety of mammalian cells.