Extensive researches are going on to explore the effective and safe drug for their hair growth. Tobacco leaves are traditionally known to potentiate hair growth promotion. Therefore, the aim of present study was to formulate and evaluate the microbial biotransformed extract of tobacco leaves for hair growth potential in male albino wister rats.The extract of was prepared by microbial biotransformation of tobacco leaves in cow urine for 28 days. The herbal formulations (lotion) were formulated by general method using o/w type base in various rations or concentrations such as 10%, 20% and 30% of extract. These lotions were applied on shaved skin area of rats for 30 days once in a day and hair length, serum total protein, and total testosterone were measured.Our formulations show increase in hair growth and serum total protein at concentration dependent manner with effect to standard and control groups. Serum total testosterone decreases according to a concentration dependent manner.Further, series of investigations are, however, necessary to remain exploration, which includes their structural elucidation, characterization, clinical safety, reliability and molecular mechanism involved in this pharmacological activity.