Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase (MAPK) kinase kinase family and elicits a wide variety of cellular responses to various types of stress through activation of the JNK and p38 MAPK pathways. ASK1 is preferentially activated in response to oxidative stress, but this regulatory mechanism is still not completely understood. In our previous report, thioredoxin (Trx), which is an antioxidant protein and plays pivotal roles in maintaining intracellular redox balance, inhibited ASK1 kinase activity by direct binding to ASK1 under normal conditions. Under oxidative conditions, ASK1 is dissociated from Trx and therefore fully activated. The active site of Trx contains two cysteine residues that undergo reversible oxidation to form a disulfide bond with each other, so that the conformation of Trx is changed by intracellular redox conditions. Thus, the oxidative stress-induced conformational change of Trx is particularly important for interaction with and regulation of ASK1, and elucidation of the regulatory mechanisms of ASK1 by Trx is critical to understanding the intracellular redox signaling. In this chapter, we review the regulatory mechanisms of ASK1 activity by Trx, and describe a method for monitoring in vitro binding between Trx and ASK1 under various redox conditions. In addition, we present methods to detect the oxidative stress-induced activation of ASK1 in the cells by Western blot analysis and in vitro kinase assay. The techniques presented in this chapter will be useful for a range of investigations into intracellular redox signaling.