The interest in developing new diagnostic methods based on arrays of multiple probes to detect and type simultaneously a wide range of different infectious agents is increasing. This becomes a necessity in the case of infectious agents such as respiratory viruses that cause diseases with very similar signs and symptoms. Such tools will permit rapid and accurate diagnosis of different agents causing respiratory infection leading to the most adequate prevention and/or treatment measures. In this article a reverse‐line blot hybridization (RLB) assay for the detection of a wide range of respiratory viruses is presented and evaluated for its usefulness in routine diagnosis. This assay employs an array of 18 oligonucleotide probes immobilized on a nylon membrane. Biotin‐labeled PCR products obtained with two multiplex reverse transcription (RT)‐polymerase chain reaction (PCR) assays described previously, which allow for the detection of fourteen different groups of respiratory viruses, were hybridized to the oligonucleotide array. Detection was performed using a chemiluminescent method. The standardization of the method showed that the RLB assay could be an alternative to the nested PCR assay for enhancing the sensitivity in the detection of the amplified products, avoiding the problem of cross‐over contamination, increasing the specificity, and therefore simplifying the method. This is of main interest in laboratories with few facilities. The feasibility and accuracy of the RT‐PCR‐RLB assay for detecting respiratory viruses proves that such approach could be a first stage to develop a microarray assay for routine diagnosis of infectious diseases. J. Med. Virol. 76:256–264, 2005. © 2005 Wiley‐Liss, Inc.