In the N-end rule pathway, a set of N-terminal amino acids, called N-degrons, are recognized and ubiquitinated by the UBR proteins. Here we examined various N-end rule inhibitors to identify essential structural components of the system. Our study using in vitro biochemical assay indicated that the L-conformation and protonated α-amino group of the first residue were critical for N-degrons to properly interact with the UBR proteins. The monomeric molecules with minimum interacting motifs showed endopeptidase-resistance and better inhibitory activities than traditional dipeptide inhibitors. Collectively, our study identifies a pharmacophore of N-end rule inhibitors, which provides a structural platform to improve the efficiency and druggable properties of inhibitors. Considering that the N-end rule have been implicated in many pathophysiological processes in cells, inhibitors of this pathway, such as p-chloroamphetamine, are potentially of clinical interest in a novel aspect of action mechanisms.