Development of approach or device to allow continuous multiple measurements, such as integrating cytotoxic and genotoxic analysis, is quite appealing for study of the drug’s activity and mechanism of action or resistance. In this study, a single-cell-arrayed agarose chip system was developed to combine cell cultivation with subsequent in situ analysis of cytotoxicity and genotoxicity of the chemotherapeutic agent. The modified alkaline comet assay coupled with the Live/Dead assay was used to monitor the interstrand cross-links (ICLs) formation and the cytotoxic effects in different glioma cell lines. In addition, the ICL-induced double strand breaks (DSBs) was measured on the chip to reflect the level of ICLs indirectly. Compared with the traditional methods, the microarray agarose device offers higher throughput, reproducibility, and robustness, exhibiting good potential for high-content drug screening.