The N-end rule pathway relates the in vivo half-life of a protein with its N-terminal residue. Recent understanding of the molecular mechanism underlying N-degron recognition implies that the yeast N-degrons may not be identical to those of mammals. Here we re-evaluate the role of N-terminal amino acids as degradation determinants through in vitro degradation assay and computational docking analysis. To take advantage of the distinct binding modes of type 1 and type 2 destabilizing residues, we developed and optimized heterovalent inhibitors of the N-end rule pathway. These small-molecules effectively delayed the degradation of physiological N-end rule substrates in vitro and in living cells, including cardiomyocytes, suggesting that the heterovalent inhibitors could be applied to various cardiac diseases that originate from abnormal N-end rule regulation.