Sanitary selection and certification of olive cultivars require sensitive diagnostic methods and effective sanitation protocols. Although much attention has been paid in the past few years to the development of diagnostic tools for reliable virus identification, the need to define a common and standardized diagnostic protocol led to the implementation of a ring test among nine Italian diagnostic laboratories. A one-step RT-PCR protocol and different primer sets, targeting the most common olive viruses covered by phytosanitary rules, were tested in each laboratory, using the same batch of positive and healthy controls as well as the same amplification conditions and reaction components. The one-step RT-PCR, performed using several specific primer sets, was able efficiently to detect the target viruses in all laboratories. Furthermore, a one-step RT-PCR protocol was used successfully for the first time for detection of Tobacco necrosis virus (TNV) and Olive mild mosaic virus (OMMV). Results showed that all target viruses were not uniformly distributed in the canopy, and that at least two subsets of samples must be collected from each plant. This standardized protocol is now being used to produce nuclear stocks for 70 different Italian olive cultivars, in the framework of the national project OLVIVA, which involves 25 national research institutions.