Imported high-risk plants spend up to 2.5 years in Post Entry Quarantine (PEQ) facilities prior to their release to plant industries. During their time in PEQ, imported plants are screened for exotic pests including viruses, viroids, bacteria, fungi and nematodes. Existing post entry quarantine screening protocols rely on resource intensive molecular and serological tests and time-consuming biological indicators. The prolonged PEQ screening schedule impacts plant industries’ capability to access new national and international market opportunities. The use of next-generation sequencing (NGS) and suitable bioinformatics pipelines to sequence and analyse small RNAs arising from the natural plant antiviral response system can accelerate the screening process of plants in quarantine. This strategy offers a number of advantages over existing PEQ protocols, namely improved accuracy, scalability, reduction in PEQ costs and screening times facilitating more rapid release of new genetics. We have recently implemented an automated viral diagnosis bioinformatics toolkit using the Yabi web-based analytical environment that enables improved detection of viruses and viroids. Here we present the results from side-by-side trials for Citrus, Prunus, Fragaria, Vitis and Solanum using traditional PEQ testing protocols and small RNA NGS method. These trials were undertaken at the Department of Agriculture and Water Resources Australia and Ministry for Primary Industries New Zealand, to confirm the sensitivity, specificity and robustness of the proposed new method.