RNA viruses are a diverse group of viruses responsible for a wide range of plant diseases and a major threat to plant health. The procedure for a simple, ultrasensitive, and specific method using a reverse transcriptase-nested polymerase chain reaction is provided for the detection of RNA viruses from naturally infected plants. This protocol consists of RNA extraction, cDNA synthesis, RT-PCR, and RT-nPCR. Two sets of primer pairs were used in which the outer primers amplify a large fragment of target DNA and nested primers amplify a smaller, more specific region within the first amplicon. This is a powerful tool for amplifying RNA targets with high sensitivity and specificity and could be a promising diagnostic application.