The γH2AX assay is widely used as a marker of DNA damage in multiple scientific fields such as cancer biomarker, clinical studies and radiation biology. In particular, the in vitro γH2AX assay has been suggested as a novel in vitro genotoxicity test with potential as a pre-screening tool. However, to date, limited assessments have been carried out to evaluate the sensitivity, specificity and accuracy of the in vitro γH2AX assay. In this study, the microscopy-based system combining automated cellular image acquisition with software quantification for High Content Screening (HCS) has been used for the first time to evaluate the in vitro γH2AX assay. A panel of well-characterised genotoxic and non-genotoxic compounds was selected to assess the performance of the in vitro γH2AX assay in the human bronchial epithelial cell line BEAS-2B. The results obtained during this preliminary assessment indicate that the in vitro γH2AX assay has a high accuracy (86%) as a result of high sensitivity and specificity (86–92% and 80–88% respectively). Our data highlight the potential for γH2AX detection in HCS as a complement to the current regulatory genotoxicity battery of in vitro assays. We therefore recommend more comprehensive assessments to confirm the performance of the in vitro γH2AX assay by HCS with a more extensive set of compounds.