HslVU is the proteasome-related system composed of HslV peptidase and HslU chaperone. It is involved in intracellular proteolysis. The presence of HslVU homologs in pathogenic microbes and its absence in human makes it an antimicrobial drug target. The functional HslVU complex forms when HslV dodecamer is flanked at both ends by HslU hexamers. In the HslVU complex, intercalation of C-termini residues of HslU subunits into the clefts between adjacent HslV subunits results in allosteric activation of HslV. We identified small molecules capable of activating HslV peptidase in the absence of its natural activator HslU. Quinazoline and chromone derivatives were suggested by ligand docking to bind at the HslU C-termini intercalation pockets in the HslV. This was confirmed by HslV activation assays with these compounds that gave ED50 in sub-micromolar range. The results showed that small, extracellular non-peptidic molecules can activate the HslV peptidase which in turn would initiate intracellular proteolysis.