Standardization of herbal formulation is essential to assess drug quality and therapeutic value. Jatyadi Ghrita, a polyherbal topical preparation from Ayurvedic formulary of India, is used for wound treatment. This study develops a standardization method for some ingredients using HPTLC with Glycyrrhetinic acid, Ursolic acid, Karanjin, Curcumin, Berberine, and Kutkin as markers. A reference laboratory sample and four marketed samples were evaluated to demonstrate method applicability for Jatyadi Ghrita standardization. © 2014 Elsevier B.V., All rights reserved.