Introduction: Trikatu Churna is an Ayurvedic polyherbal formulation useful in wide range of diseases and disorders. Efficacy of formulation depends on their genuineness of herbs used. Authentication of herbs by anatomical studies is first and fundamental step for standardization of herbal formulation. In this paper Pharmacognostic investigations like macroscopic, microscopic and chemical studies like preliminary phytochemical, physico-chemical constants and TLC/HPTLC fingerprint of Trikatu Churna were studied. Methods: The standard methods recommended in Quality Control Methods for Medicinal Plant Materials by WHO, 1998 was followed. Results: Macro-microscopic, preliminary phyto-chemical studies and TLC/HPTLC studies of the formulation has been documented. Conclusion: Findings of the study is helpful in standardization of polyherbal Ayurvedic formulation Trikatu Churna, which will promote global acceptance of the formulation and reputation of the Ayurveda system.