Human enteroviruses and rhinoviruses are respiratory pathogens whose role in acute respiratory infection is underestimated due to the use of diagnostic procedures with poor sensitivity. To determine the prevalence of these two pathogens in the upper respiratory tract infections, a multiplex procedure was developed that both detect and differentiate rhinoviruses and enteroviruses. This sensitive procedure allowed the detection of both pathogens from archival material (nasal swabs) collected during the previous winters and differentiated rhinoviruses from enteroviruses. This procedure can be used to determine the role of these pathogens in mild or severe upper respiratory tract infections.