Human rhinoviruses (HRV) are the main cause of the common cold. Viral replication utilizes the activity of the HRV3C protease (3CP) enzyme [Antimicrob. Agents Chemother. 43 (1999) 2444; Antimicrob. Agents Chemother. 44 (2000) 1236]. Therefore, 3CP is an attractive target for antiviral drug development, and a new class of orally bioavailable irreversible 3CP inhibitors has been designed [J. Med. Chem. 45 (2002) 1607]. We have used related inhibitors to develop a rapid test for rhinovirus. The optical immuno assay (OIA) thin film detection technology utilizes an optically coated silicon surface to convert specific molecular binding events into visual color changes by altering the reflective properties of light through molecular thin films. The purpose of this study was to develop a rapid assay for the determination of 3CP combining the Thermo Electron Bio Star OIA technology and the newly designed inhibitor compounds. The advantage of this assay was in its approach, in which therapeutic and diagnostic targets are the same thus allowing patients with detected rhinoviruses to receive optimal treatment. Three different biotinylated inhibitor compounds were synthesized. The length of the spacer between the inhibitor and biotin core was 5, 10, and 15 atoms. These compounds were incorporated into the OIA format for the HRV assay development. A rapid (20 min) OIA test was developed using a 15 atom spacer biotinylated inhibitor (4). Forty different HRV serotypes were studied and thirty three serotypes of these 40 were detected (80%).