Background: Several reports acknowledge that herbal products produce toxicities due to their poor quality. Hence, the worldwide promotion of traditional medicines is not at par due to a lack of quality control and safety aspects. Habb-i-Hayat, a Unani formulation, is used for the treatment of various bodily ailments. Aim: The present study was aimed at evaluating the quality control and acute toxicity of Habb-i-Hayat. Materials and Methods: The standardization and quality control study of Habb-i-Hayat, including organoleptic characters, physicochemical properties, qualitative phytochemical evaluation, microbial load, aflatoxins and heavy metals analysis, and HPTLC, were carried out. The acute toxicity study of the same was carried out at three dose levels following OECD guidelines 423. Results: The quality control findings of Habb-i-Hayat, including by weight variation, hardness, friability, moisture content, ash values, swelling index, etc., were found within the normal limits. The TBC and TFC were found within the permissible limits. The heavy metal analysis report showed that the heavy metals, including lead, cadmium, arsenic, and mercury, were present within the permissible limits. The number of peaks, Rf values, and area under the curve found in the HPTLC study may be used for identification and quality of Habb-i- Hayat. The toxicity data of Habb-i-Hayat revealed that it produces some serious toxic control effects including death at higher doses. The LD50 of Habb-i-Hayat was found to be 2500 mg/kg b. w. for rats. Conclusion: The standardization and quality control reports of Habb-i-Hayat have suggested that the preparation, drying, and storage of Habb-i-Hayat followed standard operating procedures. Moreover, the findings of standardization and quality control of Habb-i-Hayat may be referred in future. The acute toxicity profile of Habb-i-Hayat has confirmed that this important Unani formulation may be safely used at therapeutic dose levels.