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The cost for high-throughput sequencing (HTS) has decreased significantly and has made it possible for the application of this technology for routine plant diagnostics. There are constraints to the use of HTS as a diagnostic tool, including the need for dedicated personnel with a bioinformatic background for data analysis and the lack of a standardized analysis pipeline that makes evaluating and validating results generated at different HTS laboratories difficult. E-probe diagnostic nucleic acid analysis (EDNA) is an in-silico bioinformatic tool that utilizes short curated electronic probes (e-probes) designed from pathogen-specific sequences that allow users to detect and identify single or multiple pathogens of interest in raw HTS data sets. This platform streamlines the bioinformatic data analysis into a graphical user interface as a plant diagnostic tool used by diagnosticians. In this study, we describe the process for the development, validation, and use of e-probes for detection and identification of a wide range of taxonomically unique citrus pathogens that include citrus exocortis viroid, citrus tristeza virus, ‘ Candidatus Liberibacter asiaticus’, and Spiroplasma citri. We demonstrate the process for evaluating the analytical and diagnostic sensitivity and specificity metrics of the in-silico EDNA assays. In addition, we show the importance of including background noise (internal controls) to generate variance in noninfected samples for a valid statistical test using the quadratic discriminant analysis. The fully validated EDNA assays from this study can be readily integrated into existing citrus testing programs that utilize HTS. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .