Lawsonia inermis (henna) is one of the most effective medicinal plants and it has been using for treatment of wounds and burns for centuries. The using of Henna leaves is very popular for cosmetic as well as medicine in many countries. Henna leaves contain lots of different compounds and lawsone (LW) is the main one. In current study, extraction with bidistillated water of henna leaves was performed and LW was isolated by using high performance liquid chromatography system. Chemical structure of LW was evaluated by nuclear magnetic resonance method. LW was radiolabeled with iodine-131 (I-131) radionuclide which is well known for nuclear imaging and therapy in nuclear medicine by utilizing iodogen method. The yield of radiolabeling of LW (I-131-LW) was calculated as 92.70 +/- A 4.312 % (n = 10) by thin layer radio chromatography. Its in vivo biological activity was investigated by biodistribution studies which were performed by using healthy female and male Balb/C mice. According to results of biodistribution, uptake of I-131 labeled LW compound in uterus, breast and ovary for female mice and prostate in male mice was higher than other organs in the body.