To enhance the affinity of peptide ligands for their targets, covalent warheads can be engineered to facilitate irreversible binding. This study aimed at exploring the potential of a 68Ga-labeled peptidomimetic radioligand, [68Ga]Ga-DOTA-RQAR-kbt, for PET imaging through its irreversible binding to the suppression of tumorigenicity 14 (ST14). An Arg-Gln-Ala-Arg (RQAR) tetrapeptide was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid for gallium-68 radiolabeling. The covalent warhead ketobenzothiazole was constructed as a serine trap for ST14 protease, resulting in the formation of DOTA-RQAR-kbt. We compared both the in vitro and in vivo properties of [68Ga]Ga-DOTA-RQAR-kbt with those of its reversible-binding counterparts, [68Ga]Ga-DOTA-RQAR-OH. DOTA-RQAR-kbt exhibits high affinity for ST14 and irreversibly binds to ST14, as evidenced by the lack of ST14 activity recovery following ultrafiltration. In contrast, DOTA-RQAR-OH shows reversible binding and has low affinity for ST14. PET/CT imaging confirmed the superior tumor targeting of [68Ga]Ga-DOTA-RQAR-kbt compared to the [68Ga]Ga-DOTA-RQAR-OH, with robust signals observed at 0.5, 1, and 2 h postinjection. Blocking studies underscored the probe's specificity, as they revealed a marked reduction in tumor uptake in the presence of excess RQAR-kbt. Biodistribution studies demonstrated significantly higher tumor uptake for [68Ga]Ga-DOTA-RQAR-kbt, with 0.89 ± 0.03%ID/g at 1 h postinjection, which was reduced to 0.25 ± 0.03%ID/g (P < 0.01) in the presence of excess RQAR-kbt. In this proof-of-concept study, an irreversibly binding peptidomimetic radioligand targeting ST14 was evaluated, demonstrating improved tumor uptake in vivo compared with its reversibly binding counterparts. This approach holds promise for improving the potency of covalent radiotracers as PET agents.