The control of intracellular protein homeostasis is essential for the ability of plants to grow under different physiological conditions, as well as respond to various biotic or abiotic stresses. One of the ways that cells achieve this equilibrium is through the targeted proteolysis of proteins by the ubiquitin-proteasome system. A subset of this system, termed the N-end rule pathway, relates the in vivo longevity of a substrate protein to the nature of its N-terminal amino acid. Although the N-end rule pathway is known to regulate numerous physiological processes in plants relatively few substrates of the pathway have been identified to date. In this study experiments were conducted aimed at identifying N-end rule substrates in the model plant Arabidopsis thaliana. One group of candidate substrates is generated after their proteolytic cleavage by a bacterial effector protein. The transient expression of these candidate N-end rule substrates in tobacco coupled with pathogen inoculation and biochemical methods led to the identification of a group of protein fragments that are likely novel N-end rule substrates. Experiments were also conducted towards developing a molecular tagging tool with the aim of conducting a proteome-wide screen for N-end rule substrates. Additionally, experiments were carried out to characterize a component of the Arabidopsis N-end rule pathway by expressing this enzyme in the yeast S. cerevisiae under different conditions. This study will allow for a further understanding of the involvement of the N-end rule pathway in plant-pathogen interactions and provides several novel N-end rule substrates for future experiments aimed at dissecting the diverse functions of this pathway in plants.