<p>Grapevine (<i>Vitis</i> spp.) and other vegetatively propagated plants are affected by numerous viral diseases, often leading to serious pathological problems. In recent years, specific and rapid diagnostic methods have become increasingly important for selecting propagation materials. Symptoms such as flecking, stunting, red blotch disease, and rugose wood are associated with several grapevine-infecting viruses. These include grapevine asteroid mosaic-associated virus, grapevine Pinot gris virus, grapevine red blotch virus, grapevine red globe virus, grapevine rupestris vein feathering virus, grapevine Syrah virus 1, and grapevine virus D. However, these viruses are difficult to detect based on symptoms alone. Therefore, a two-step reverse transcription (RT)-multiplex polymerase chain reaction (PCR) assay was developed as a rapid and cost-effective method for the simultaneous detection of these seven viruses using newly designed primer sets. Primers were designed for each viral variant based on sequence data obtained from the National Center for Biotechnology Information and were thoroughly evaluated in silico. The newly developed RT-multiplex PCR assay enabled the simultaneous detection of all seven viruses. No clear differences in detection limits were observed between the developed assay and previously reported RT-conventional PCR assays. In addition, almost no non-specific reactions occurred. Furthermore, differences in viral concentrations did not significantly affect detection performance, and all viruses were successfully detected. The proposed assay will be useful for identification and selection of healthy grapevine planting materials.</p>