Plants produce a wide range of active principles, making them a rich source of different types of medicines. Without any knowledge of the phytoconstituents or active principles the medicinal plants itself or in the form of polyherbal formulations, were used by all society of human being from ancient times for prevention and cure of disease, but most of the traditional formulations including the formulation of Ayurveda and Unani have not been scientifically validated in order to establish the pharmacopoeial standards to improve the efficacy. Globally, the people become conscious that uses of synthetic drugs for a long period is not safe; the trend of medical society at large is looking at alternatives from natural, safe sources to combat diseases. Due to this comprehension, it has been increased the demand for plant-derived medicine, and on the other side, it is extremely important to standardize the polyherbal formulations and validate them scientifically to improve their safety and efficacy. The polyherbal Unani formulation Safuf-e-Muallif is widely used and recommended in Unani system of medicine (USM) as a spermatogenic agent, semen thickening agent, etc. to treat sexual disorders viz. premature ejaculation, nocturnal emission, etc. The study mainly deals with phytochemical screening for the detection of nature of phytoconstituents and analytical technique like High-performance thin-layer chromatography (HPTLC) for developing fingerprint of Safuf-e-Muallif revealing specific identities of the drug. The phytochemical screening and HPTLC fingerprint profile for SM reported here may be used as a reference standard for quality control purpose in future.