The traditional systems of medicine are being subjected to rigours of standardization to compete with national and international markets to establish their credibility. The present paper involves an Ayurvedic formulation Rasayana churna. It comprises of the dried powders of three rejuvenating drugs viz., dried stem of Guduchi (Tinospora cordifolia Miers.), dried fruit of Gokshur (Tribulus terrestris Linn.) and dried pericarp of Amalaki (Emblica officinalis Gaertn.). All these plants are used in rasayana therapy. Rasayana Churna plays a very important role as an immunomodulator, rejuvenator, adaptogen, antioxidant and diuretic. Other important roles of rasayana churna involve elimination of toxins through urinary system and lowering the risk in kidney and cardiac disorders. The present study aims at analysis of the evaluation parameters of the in-house and two marketed samples of rasayana churna (coded as IHRC, MRC and ZRC), for its morphological, microscopical, phytochemical, physicochemical, spectroscopical and biological evaluation aspects. The need of standardisation is justified in the range of variations obtained in the results of morphology and physicochemical parameters. The highest berberine content was found in MRC (1.19 μg/mL). The highest phenolic content was found in MRC (64.29±2.91 mg GAE/g) and highest flavonoid content was found in MRC (18.99±0.40 mg QUE/g). Samples also showed antioxidant activity in correlation with the amount of phenolic and flavonoid content present in them (MRC had the maximum activity with IC50value of 204.09±14.64 μg/mL and 8.18±0.65 μg/mL for H2O2 and NO free radicals). The antiurolithiatic activity was studied and was found to be maximum for the aqueous extract of ZRC with 47.15% inhibition at 100 μg/mL within the first 2 min.