The peach tree “Prunus persica” is widely cultivated in India, and its fruits which have been used for centuries in traditional Indian medicine, are also known as the Queen of the Fruits. Prunus persica has immense therapeutic potential, widespread use in folklore and ancient systems of medicine for its anti-urolithic and other medicinal properties, and is included in the Pashanbheda group of herbs mentioned in Ayurveda for the treatment of kidney stones. Moreover, with growing interest in phytopharmaceuticals in the present world, there always remains an urge for a comprehensive understanding of the phytochemicals responsible for the medicinal activity of herbal drugs, giving rise to the importance of conducting bioassay-guided fractionation. In this study, the anti-urolithiasis activity of different fractions of Prunus persica fruit extracts was evaluated via Nucleation, Aggregation, and Growth assays, and fraction III showed the highest effects in vitro. This study provides a pharmacological basis for using Prunus persica fruit extract in treating kidney stones. Manuscript profile