Detection of respiratory virus in veterinary species has traditionally relied on circulating antibody detection methods such as ELISA and virus isolation. However, multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is a complex infectious disease and one of the most common causes of morbidity and mortality in housed cattle. A one-step multiplex real time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) in clinical samples from cattle. The assay detects a region encoding a highly conserved glycoprotein B gene of BoHV-1, nucleocapsid region of BRSV and nucleoprotein region of BPI3. This multiplex real time RT-PCR assay was validated for sensitivity, specificity and repeatability using clinical isolates and samples from naturally infected animals and compared with results from virus isolation and fluorescent antibody tests (FAT). The multiplex assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 10^2 copies of BRSV, BoHV-1 and BPI3 i & ii (Cp 34 to 35). This is the first one-step multiplex real time RT-PCR developed to detect four major aetiological agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binder (MGB) and taqman probes in one reaction mix. This test is more sensitive than virus isolation and FAT and therefore, better disease diagnosis during outbreaks.