Lawsone, also known as hennotannic acid, is the natural pigment obtained from henna. Lawsone is chemically 2-hydroxy-1,4-naphthoquinone. The presence of the hydroxyl group is the major determinant of its biological activity. Lawsone is extracted from the henna plant by solvent extraction using nonpolar solvents. Column chromatography is mainly used for the isolation of lawsone from the extract. High Performance Liquid Chromatography, coupled with spectroscopic detectors, is employed for analytical quantification. Lawsone possesses anti-cancer, anti-bacterial, anti-fungal, molluscicidal, anti-parasitic, hepatoprotective, wound healing, anti-inflammatory and antiplatelet activities. It reduces the proliferation of DLD-1 cells via the NF-kB pathway in colon cancer cells. Lawsone is effective against skin cancer. Lawsone has antibacterial activity against different types of bacteria including multidrug-resistant Staphylococcus aureus. The antioxidant potential of lawsone is the reason for its hepatoprotective effect. This review deals with the isolation, quantification, and pharmacologic activities of lawsone.