In traditional Indian medicinal treatise there are several Ayurvedic formulations mentioned which have been claimed as potential wound healing agents like Madhu Ghrita and Jatyadi ghrita. Jatyadi ghrita (JG) is a medicated ghee formulation popularly used in the treatment of various topical wounds. It is a polyherbal preparation containing eleven ingredients. Though JG has its composition recorded in ancient Ayurvedic texts, there have been minimal attempts to standardize its. In this research paper an attempt has been made to develop standardization method for some of the ingredients of Jatyadi Ghrita.Quantitative estimation was done by reported RP-HPLC methods using markers glycyrrhetinic acid, ursolic acid, karanjin, curcumine, berberine and kutkin which are major constituents of formulation. A standard laboratory reference sample of Jatyadi Ghrita and four marketed samples were evaluated as per methods. Data has been provided to demonstrate applicability of the methods to standardization of Jatyadi Ghrita. © 2013 Elsevier B.V., All rights reserved.