Introduction: Root of Aristolochia indica Linn. has long been used as an oxytoxic agent to aid women in child birth and as abortifacient in Indian folk medicine. It is also one of the ingredients in some traditional Ayurveda medicinal preparations. Aims: The present work has been designed to delineate the pharmacognostic profile of the root of Aristolochia indica Linn and the High-performance thin-layer chromatographic (HPTLC) identification of the active compound and its quantitative estimation in the herbal sample. Materials and Methods: Macroscopic, microscopic evaluation, powder analysis, fluorescence standards of the root of Aristolochia indica Linn and its HPTLC fingerprint profile.Results: Pharmacognostic profile of the root investigated revealed the transverse section possessing somewhat circular outline with tissue organization as outer thin walled cork layers, narrow cortex, and inner cortical cells with groups of stone cells. Secondary xylem tissues were fissured to form narrow strips, wide medullary rays with greater quantities of parenchyma, ray cells with rich deposition of starch. Vessels were solitary and occluded with tyloses and starch grains with 'Maltese cross' were the characteristic features of the taxon. HPTLC method was developed for the estimation of the marker constituent, Aristolochic Acid I (AAI) in dried root sample. Chloroform: Methanol (6:2v/v), was used as mobile phase to separate the analyte. The Rf value for Aristolochic Acid I (C17H11NO7) was found to be 0.53. Calibration plot was established showing the dependence of