Background Several bacterial and viruses can infect the respiratory tract of small ruminants causing similar clinical signs. The differential diagnosis of respiratory diseases in small ruminants can be achieved using multi-plex assays for an accurate identification of the causative agents. Objective The present study was aimed at developing molecular multiplex as-says using different methods, such as real time PCR and micro fluidics bead-based technology, applicable for the syndromic surveillance of respiratory infection in small ruminants. The targeted infections were those caused by Capripoxviruses (CaPVs), Peste-des-petits ru-minants' virus (PPRV), Parapoxvirus, Mycoplasma Capricolum subsp. Capripneumoniae (MCCP) and Pasteurella multocida (PM). An inter-nal control was included in order to determine the quality of sam-ples being tested. Methodology Primers and probes were designed for the conserved regions of the genomes of all the targeted pathogens. The probes for real time PCR were labelled with compatible fluorescent dyes and quenchers, whereas for microfluidics bead based method, primers and probes were biotinylated, phosphorylated and C12 amino-modified accor-dingly. Total nucleic acid extraction procedures were evaluated to extract both DNA or RNA. The amplification protocols were opti-mized and the procedures were validated for the amplification of the above-mentioned pathogens in a single test (or tube). Results A one-step multiplex real time PCR method was developed to ampli-fy four targets, CaPVs, PPRV, MCCP and PM in order to accommodate real time PCR platforms from different manufacturers and reduce complexity in performing the assay. This real time PCR method was highly specific and sensitive in detecting the targeted pathogens as well as co-coinfections. Out of 314 samples tested from different African countries, 80 samples were positive for PPRV, 50 for PM, 2 for CaPV and 8 were mixed infections of PPRV and PM. The same patho-gens were included, a