This study evaluated a single-tube multiplex RT-PCR with a primer focusing on nonstructural protein region 5 (NS5) and a highly conserved region for dengue virus serotyping. The method was compared with conventional PCR. This new method had a sensitivity of 96.7% and a specificity of 96.7% for disease detection. The new method also proved suitable for use in the field as it reduces time and decreases risk of contamination.