Background Pashanabheda is an important Ayurvedic drug known for diuretic and lithotriptic properties. The botanical identity of this drug is controversial as many plants like Bergenia ciliata (BC), Rotula aquatica (RA), Aerva lanata (AL) and Plectranthus amboinicus (PA) are being used as its source plants in different parts of India. This study aims at comparative phytochemical and pharmacological evaluation of various source plants of Pashanabheda. Methods Comparative phytochemical analysis and evaluation of Antiurolithiatic activity was carried out in selected medicinal plants used as Pashanabheda. Chemical profiling was done by HPTLC analysis and characterization of major constituents was done using liquid chromatography based tandem mass spectroscopic analysis. Antiurolithiatic activity was evaluated by ethylene glycol induced urolithiasis in experimental animal model. Results Phytochemical studies showed that there is no significant correlation on chemical constituents of selected species. Certain common bands were observed for BC and RA on HPTLC profiling. Tandem mass spectroscopic characterization of various species showed presence of several polyphenolics in selected species. Both Bergenia ciliata and A. lanata possessed substantial antiurolithiatic activity compared to other species. A. lanata extract at doses of 100 and 200 mg/kg, showed significant activity against ethylene glycol induced changes serum and urine biochemistry and also significantly prevented ethylene glycol induced damage to nephrons in experimental animals. Conclusions Aerva lanata contains many active phytochemicals and showed significant antiurolithiatic activity. The findings of this study may lead to the possibility of considering Aerva lanata as a validated alternative source for Pashanabheda.