Panchatikta is an Ayurvedic multiherbal combination containing five bitter‐tasting drugs—Azadirachta indica, Tinospora cordifolia, Justicia adhatoda, Solanum virginianum, and Trichosanthes dioica. The objective of this study is to develop standards for Panchatikta ingredients using pharmacognostic parameters and to analyze the chemical composition. The plant materials were collected from different natural habitats, authenticated, dried, ground, and the combination was prepared by mixing the ingredients in equal amounts followed by macroscopic, organoleptic, microscopic, histochemical, and physicochemical characterization according to the standard protocol of the Ayurvedic Pharmacopoeia of India. Special morphological characteristics of the A. indica (stem bark), T. cordifolia (stem), J. adhatoda (roots), S. virginainum (roots), and T. dioica (aerial parts) were identified. The anatomical structure of each drug was observed, and microscopic markers were developed for identification. Histochemical deposits such as starch, oil, lignin, and tannin were located in each plant material. Powder characters such as fibers from phloem with pointed ends and narrow lumen, parenchyma cells containing starch grains, crystal fibers, fragments of bordered pitted vessels, fragments of pitted vessels, cells containing black powdery masses, and epidermal cells from leaves in surface view with an anomocytic stomata were identified as the key features for the Panchatikta combination. The organoleptic characterization of component drugs and combinations was performed. The physicochemical evaluation of parameters such as ash content (5.53 ± 0.29), water‐insoluble ash (4.57 ± 0.32), acid‐insoluble ash (1.55 ± 0.31), alcohol‐soluble extract (5.9 ± 1.21), water‐soluble extract (9.19 ± 0.21), fiber content (33.27 ± 1.37), total sugar (1.83 ± 0.65), and reducing sugar (1.73 ± 0.40) were evaluated for the Panchatikta combination. Preliminary phytochemical tests were carried out to verify the presence of alkaloids, flavonoids, saponins, tannins, phenols, glycosides, and steroids. The HPTLC profile comparison of ethanolic, hydroalcoholic, and aqueous extracts of Panchatikta and single drugs showed some type of synergy and antagonistic activities among the chemical principles present. Marker‐based screening of azadirachtin, betulinic acid, and lupeol was done in Panchatikta ethanol using HPTLC. As Panchatikta is prescribed for various life‐threatening diseases including cancer, the standards developed here ensure the authenticity of the ingredients, which in turn ensures the quality of the prepared medicines.