Human tissue transglutaminase (hTG2) is a multifunctional enzyme. It is primarily known for its calcium-dependent transamidation activity that leads to formation of an isopeptide bond between glutamine and lysine residues found on the surface of proteins, but it is also a GTP binding protein. Overexpression and unregulated hTG2 activity have been associated with numerous human diseases, including cancer stem cell survival and metastatic phenotype. Herein, we present a series of targeted covalent inhibitors (TCIs) based on our previously reported Cbz-Lys scaffold. From this structure-activity relationship (SAR) study, novel irreversible inhibitors were identified that block the transamidation activity of hTG2 and allosterically abolish its GTP binding ability with a high degree of selectivity and efficiency (kinact/KI > 105 M-1 min-1). One optimized inhibitor (VA4) was also shown to inhibit epidermal cancer stem cell invasion with an EC50 of 3.9 μM, representing a significant improvement over our previously reported "hit" NC9.