Homologous recombination (HR) is a major pathway to repair DNA double-strand breaks. Hereditary breast and ovarian cancer syndrome (HBOC) is caused by germline pathogenic variants of HR-related genes, such as BRCA1 and BRCA2 (BRCA1/2). Cancer cells with HR deficiency are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Therefore, accurate evaluation of HR activity is helpful to diagnose HBOC and predict the effects of PARP inhibitors. The direct-repeat GFP (DR-GFP) assay has been utilized to evaluate cellular HR activity. However, evaluation by the DR-GFP assay tends to be qualitative and requires the establishment of stable cell lines. Therefore, we developed an assay to quantitatively measure HR activity called Assay for Site-Specific HR Activity (ASHRA), which can be performed by transiently transfecting two plasmids. In ASHRA, we use Cas9 endonuclease to create DNA double-strand breaks at specific sites in the genome, enabling the targeting of any endogenous loci. Quantification of HR products by real-time PCR using genomic DNA allows HR activity evaluated at the DNA level. Thus, ASHRA is an easy and quantitative method to evaluate HR activity at any genomic locus in various samples. Here, we present the protocols for adherent cells, suspension cells, and tumor tissues.