DNA damage response (DDR) pathways form an integral part of the body's repair machinery, and ATR (ataxia-telangiectasia and Rad3-related kinase) protein is one of the key mediators in the DDR pathway that helps in maintaining genomic integrity. A growing body of evidence suggests that inhibition of ATR can help sensitize tumor cells to combinatorial treatment. However, specific ATR kinase inhibitors have largely remained elusive until now. Despite much interest in the protein for more than a decade, there has been little characterization of only the kinase domain, an essential target site for a variety of ATR inhibitors. Here, we report our findings for the bacterial expression, purification, and biological characterization of this potentially important recombinant kinase domain, which could further be considered for structure elucidation studies. Introduction of a solubility partner, i.e., maltose binding protein (MBP), at the N-terminus of the ATR kinase domain generated a soluble form of the protein, i.e., MBP-tagged hATR kinase domain (MBP-ATR-6X His), which was found to be catalytically active, as assessed by substrate p53 Ser-15 phosphorylation (EPPLSQEAFADLWKK). Our results also highlight the prospect of utilization of the overexpressed recombinant ATR kinase domain in characterization of kinase domain specific inhibitors.