Background: Atalantia monophylla DC. (Syn. Limonia monophylla L.), family Rutaceae, root and stem have been reported for their use in rheumatism, arthritis, antidote to snake-bite, cough, allergies, skin diseases and as an antidote to snake-bite through ethnomedicinal claims. Present study reports a details pharmacognostic and preliminary physiochemical and phytochemical aspects of A. monophylla root and stem. Material and methods: The root and stem samples were collected after proper authentication from Athagarh forest Odisha India. Macroscopic observations were made with naked eyes, microscopy, powder microscopy, physicochemical and qualitative analysis and HPTLC studies of root and stem were carried out following Ayurvedic Pharmacopoeia of India recommended and others standard protocols. Results and Discussion: Root were 5-7cm in length; flattened, L-shaped, easily peel-able and thick, outer yellowish brown and inner yellow in colour with rootlets and scars. Stem cylindrical, striated, greenish brown in colour. Diagrammatic transverse section of root and stem shows circular in shape, outer cork, cortex followed by central stellar region and circular in nature, outer epidermis followed by hypodermis, cortex, vascular bundle and centrally located large pith respectively. Microscopic powder character of root and stem shows prismatic crystal, parenchyma cell with oil globule, simple starch grains, brown content and rhomboidal crystal, pitted parenchyma cells, brown content, oil globule, simple and compound starch grains respectively. Qualitative phytochemical analysis of root and stem shows the presence of carbohydrates, glycosides, saponins, alkaloids, tannins and flavonoids. HPTLC study of root and stem shows 22 and 25 spots at short (254nm) and 24 and 27 spots at long (366nm) ultraviolet respectively. Conclusion: The results obtained from pharmacognostical, phytochemical, physicochemical characteristic along with HPTLC studies are the diagnostic tool of A. monophylla plant and will help in standardization of its root and stem in terms of its quality, strength, purity.