RNA sequencing (RNA-seq) has revolutionized plant virus detection. This powerful technique offers an unbiased approach to identify a wide range of viruses, including those previously unknown. Unlike traditional methods targeting specific pathogens, RNA-seq analyzes the entire pool of RNA molecules within a plant sample. This comprehensive approach allows for the simultaneous detection of multiple viruses, even in mixed infections and also collects the host transcriptome data. In RNA-Seq experiment, removal of ribosomal RNA (rRNA) to enrich for viral RNA sequences is critical as these are overshadowed with mRNA. However, RNA-seq can detect viruses with varying genome types, including those with and without polyadenylated tails. Beyond detection, RNA-seq has the ability to not only identify viruses but also to explore host plant responses to infection through analysis of the host transcriptome data obtained along with viral sequences. This method briefly explains the careful depletion of ribosomal RNA (Sd rRNA) followed by RNA sequencing library preparation to detect a wide range of viruses.