Urostone is an innovative treatment approach for urolithiasis, a condition characterized by kidney stone formation. With a global prevalence of about 13%, urolithiasis has become a growing urological disorder in recent decades. Currently, surgical procedures are the primary treatment option and often have a high chance of recurrence. Interest in herbal drugs with low toxicity and minimal side effects has been increasing. Urostone is a polyherbal formulation comprising three plant species: Hydrangea sp, Berberis sp, and Hemidesmus sp, which aim to address the challenges associated with urolithiasis management. The objective of the study was to investigate the effectiveness of Urostone in urolithiasis treatment, including stone disintegration and facilitating stone passage. Bioactive phytochemicals responsible for the antilithiatic effects of Urostone were identified and were then tested for their binding affinity with Adenine phosphoribosyltransferase (APRT) gene and calcium-sensing receptor, as these are known to play a role in urolithiasis through calcium oxalate/phosphate accumulation and APRT gene mutation. Out of 54 ligands tested, 10 showed promising activity against the APRT gene and calcium-sensing receptors. Cytotoxicity tests using vero cells determined the inhibitory concentration (IC50) value, with an optimal value of 72 mg/ml. In animal models with induced urolithiasis, Urostone demonstrated better results compared to the control agent. Urostone could be the first proven polyherbal drug of choice for treating urolithiasis. Its comprehensive formulation, synergistic herbal ingredients, and positive results in various aspects of urolithiasis management make it a promising alternative to surgery with the potential for reduced recurrence rates and improved patient outcomes.