A substantial and low cost DNA isolation protocol is the prerequisite of many molecular biology experiments for the genetic improvement of highly economical medicinal plants. The present study was undertaken to explore the best DNA isolation protocol for Stevia rebaudiana Bert., an artificial sweetener. Four methods were evaluated for extraction of DNA namely Kit GeNeiTM Genomic DNA extraction (Bangalore GeNei), Kit GeNeiTM Plant DNA isolation (Bangalore GeNei), Phenol-Chloroform Method and CTAB method. All four methods evaluated effectively to isolate the DNA from the young and old leaves of Stevia plant and produced consistently positive results. The quantity and quality of the DNA extracted was compared using UV-spectrophotometer and agarose gel electrophoresis. It was found that the Kit GeNeiTM Plant DNA isolation (Bangalore GeNei) had good quality as well as quantity (A260/280, 1.5655) of DNA as compared to other three methods. The present findings will certainly play a vital role in genetic improvement of Stevia rebaudiana Bert.