Human High Temperature requirement A2 (HtrA2) also known as Omi, is a serine protease located in the mitochondria with an important function in both cell survival and death. My results show the proteolytic activity of Omi/HtrA2 varies under different conditions. I characterized the optimal condition for Omi/HtrA2 protease activity using an in vitro assay system. Additionally, I identified a new allosteric regulation of Omi/HtrA2 through interaction with a specific substrate, the MUL1 protein. MUL1 is a multifunctional E3 ubiquitin ligase anchored in the outer mitochondrial membrane with domains both inside mitochondria and in the cytoplasm. The data shown here strongly supports the hypothesis that Omi/HtrA2 activity is modulated by a number of different mechanisms. Some of these conditions, such as pH or substrate denaturation might reflect the state of mitochondria under stress. It has been known that Omi/HtrA2 is a stress activated protease, but the mechanism of its regulation has not been fully elucidated. Furthermore, the allosteric regulation of Omi/HtrA2 by specific substrates, can be another mechanism that provides a feedback loop to increase the activity of the enzyme. The findings from this project contribute new information on the mechanisms of activation of Omi/HtrA2 protease. They support the hypothesis that mitochondrial stress might be involved in the regulation of Omi/HtrA2 protease.