Two small libraries of tripeptidic-based vinyl ester derivative proteasome inhibitors were synthesized and tested, starting with the Hmb-Val-Gln-Leu-VE prototype. The P3 and P4 positions were investigated with a complete set of amino acid residues, some of which showed remarkable selective inhibition of the trypsin-like (beta2) subunit. In both positions, aromatic and hydrophobic residues were preferred.