The degradation of RNA is main hindrance for dealing with fine quality RNA for its downstream processing. Mostly RNAs are degraded as tissues are taken from fields, therefore the protocol for PSTVd RNA isolation was optimized and improved for obtaining Pure and high quality RNA having an increased and nondegradable life from infected plants of Potatoes. The present study was aimed to confirm the presence and identification of potato spindle tuber viroid (PSTVd) on potato plants in Pakistan. RNA was extracted and purified using optimized Trizol reagent and RNeasy Miniplant Kit. The RNA obtained was converted into cDNA on the same day and amplified. The identification of PSTVd was carried out by Sequencing and BLAST. The advanced optimized Rapid Trizol reagent method was comparatively found best and reproducible as compared to the second method. The Sequencing and BLAST confirmed the presence of Pakistani PSTVd (Accession # MK.303578) in infected potato plants.