Potato spindle tuber viroid (PSTVd) is a quarantine pathogen in the European Union and causes damaging diseases of solanaceous crops. Under the EU Plant Health directive 2000/29/EC, countries must have the ability to detect and identify accurately and rapidly the introduction of harmful organisms in plants or plant products; furthermore, if the quarantine pathogen is found, be able to survey extensively for it. In this respect, PSTVd poses an interesting technical problem, since its RNA does not code for any proteins and thus any diagnostic method must be based on the detection of the RNA and be suitable for scaling up to testing large sample numbers. With this in mind a one-tube real-time RT-PCR assay based on TaqMan chemistry was developed. Investigations were carried out into various aspects of the assay relevant to the efficient amplification of targets that have a significant amount of secondary structure such as viroids. Thus comparisons were made of reverse transcription temperature, concentration and type of reverse transcriptase, RNA denaturation, sample purity and single versus two-tube reaction format. The assay developed was shown to be able to detect a wide range of isolates of PSTVd and in comparison with a chemi-luminescent hybridisation system was shown to be 1000-fold more sensitive. A further significant advantage of this assay format compared with hybridisation is that it is suitable for scaling up to large sample numbers using robotic liquid handling systems.