Human rhinovirus encoded 3C protease is an attractive target for antiviral drug development. However, lack of a convenient and selective assay for 3C protease has been a hindrance in characterization of this enzyme and evaluation of a large number of potential inhibitors. In the present study we describe development of a simple, continuous colorimetric assay for this enzyme using peptide p-nitroanilides (pNA) as substrates. Several peptides mimicking the native 3C cleavage site of HRV-14 polyprotein have been synthesized with an N-acylated p-nitroaniline at position P1' and examined as substrates for the purified 3C protease. In these peptides, amino acids downstream from the original cleavage site have all been replaced with a chromophoric p-nitroaniline moiety which is directly linked to the bond undergoing enzymatic cleavage, thereby generating a new cleavage site Gln-pNA for the enzyme. Hydrolysis of these pNA peptides by 3C at the newly formed scissile bond releases free p-nitroaniline which is yellow-colored and can be continuously monitored at a visible wavelength. Kinetic parameters of 3C protease toward these peptides have been measured and analyzed. In addition, the pNA peptides have been modeled within the active site of the 3C protease to investigate the ability of the pNA group to act as a replacement for Gly-Pro in the prime side. The selectivity and applicability of this assay and its advantages over the previously described methods have been demonstrated and discussed. Since multiple tests can be performed simultaneously in one microtiter plate, the assay is ideal for evaluation of a large number of samples.