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Background: Though a number of studies has been carried out in terms of safety of Kushta but concerns have been raised about the presence of heavy metals in Unani formulations. Classical method of preparation of Kushta Khabasul Hadeed (KKH) and other Kushta preparation in Unani system of medicine has been found to eliminate all such issues from the end product. However, there is a need to provide scientific evidence to establish the impact of the procedure, hence; the present study was designed to evaluate the acute and sub-acute toxicity study of KKH prepared by Classical and Muffle furnace methods. Objective: The investigators in this study made an attempt to ascertain the safety profile of Kushta Khabasul Hadeed (KKH) prepared by classical and muffle furnace methods. Materials and methods: Both of the samples of KKH i.e. Kushta Khabasul Hadeed (KKHCM) and Kushta Khabasul Hadeed (KKHMFM) prepared by Classical and Muffle Furnace Methods respectively were evaluated for acute and sub-acute toxicity by OECD guidelines 423 and 407 respectively. The starting dose in the acute test was 50 mg/kg and in sub-acute test, it was 134mg, 200 mg and 400 mg/kg b.w. Effect of both of the test samples were assessed for ponderal changes, food and water intake, relative organs weights, hematological, biochemical and histopathological features of various organs. Results: Both of the samples did not produce any sign of toxicity in acute toxicity study up to the dose level of 2000mg/kg b.w. In sub-acute toxicity study no mortality in any of the test groups was observed. KKHCM at different doses showed a slight decrease in food intake in all the test groups but significant difference (P<0.05) was observed in the test group B as compared to plain control. However, no alteration in food intake was noted in Satellite group. Repeated dose administration of KKHMFM at different dose levels lead to a significant decrease in food intake in the test group A (P<0.01) While in the test groups B, C and satellite, the decrease was not significant in comparison to plain control. Conclusion: KKHMFM was found to be relatively toxic than KKHCM at different dose levels. The study demonstrated that both the Classical and Muffle furnace methods of preparation of Kushta are useful for preparing KKH and also safe at the dose prescribed in Unani literature. However, the Kushta prepared by the classical method was found to be safer than the muffle furnace method. [ABSTRACT FROM AUTHOR]