RUSKI-201 dihydrochloride is a potent and specific Hedgehog acyltransferase ( Hhat ) inhibitor with an IC 50 of 0.20 μM. RUSKI-201 dihydrochloride is able to block Hh signaling from Shh overexpressing cells and inhibits Hh palmitoylation. RUSKI-201 dihydrochloride is potential Hhat chemical probe in cells and can used in studies of Hhat catalytic function.