Eukaryotic cells regulate progression through the cell cycle in response to DNA damage. Cell cycle checkpoints are the signal transduction pathways that couple the detection of DNA damage to the proteins that control transitions in the cell cycle. The protein kinase Chk1, originally discovered in fission yeast, but conserved in humans, is essential for preventing mitotic entry in the presence of DNA damage or blocks to DNA replication that cannot be reconciled. Chk1 is phosphorylated in response to DNA damage. Phosphorylation depends on the activity of conserved components of the checkpoint pathway including Rad3, a member of the ATM/ATR family of kinases. Phosphorylation leads to activation of Chk1 kinase activity. In this chapter, we describe an assay for monitoring the activity of Chk1 isolated.