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Summary: The repair products of double-stranded DNA breaks (DSBs) are crucial for investigating the mechanism underlying DNA damage repair as well as evaluating the safety and efficiency of gene-editing; however, a comprehensively quantitative assay remains to be established. Here, we describe the step-by-step instructions of the primer extension-mediated sequencing (PEM-seq), followed by the framework of data processing and statistical analysis. PEM-seq presents a full spectrum of repair outcomes for both genome-editing-induced and endogenous DSBs in mouse and human cells.For complete details on the use and execution of this profile, please refer to Gan et al. (2021), Yin et al. (2019), Liu et al. (2021a), and Zhang et al. (2021).