The efficacy of a series of diazomethyl ketones (DMKs) was measured in rhinovirus-infected cultures and against the HRV14 3C protease. Their specificity and potency were confirmed against purified recombinant enzyme expressed in a yeast secretion system. An internally quenched fluorescent peptide substrate was used to assess the potency against the enzyme, obtaining a 50% inhibitory concentration (IC50) of 1 μM for both Z-L-F-Q-CHN2and Z-V-L-F-Q-CHN2, while a lower affinity was observed for Z-F-Q-CHN2. The tripeptide Z-L-F-Q-CHN2blocked viral replication with an IC50value of 30 μM as judged by the reduction in viral induced cytopathy of HeLa-H1 cells, as well as a marked reduction in viral plaque formation (50% effective concentration=20 μM). Western blot analysis of viral proteins from infected cells indicates that this inhibitor works specifically by blocking viral polyprotein maturation, displaying a reduction of detectable 3C protease and an accumulation of the 3CD polypeptide. These results indicate that DMK inhibitors of the 3C protease have antiviral potency. Furthermore, the pattern of viral protein processing observed suggests that reducing the concentration of mature HRV 3C protease even in the presence of increased 3CD protein is sufficient to block proper viral processing and significantly reduce virus yield.