The activity and abundance of proteins within a cell are controlled precisely to ensure the regulation of cellular and physiological processes. In eukaryotes, this can be achieved by targeting specific proteins for degradation by the ubiquitinproteasome system. The N-end rule pathway, a subset of the ubiquitinproteasome system, targets proteins for degradation depending on the identity of a protein N-terminal residue or its post-translational modifications. Here, we discuss the most recent findings on the diversity of N-end rule pathways. We also focus on recently found defensive functions of the N-end rule pathway in plants. We then discuss the current understanding of N-end rule substrate formation by protease cleavage. Finally, we review state-of-the-art proteomics techniques used for N-end rule substrate identification, and discuss their usefulness and limitations for the discovery of the molecular mechanisms underlying the roles of the N-end rule pathway in plants.