Gami-Samhwang-San, a herbal prescription for obesity treatment, is composed of seven crude herbs such as Rehmanniae Radix Preparata, Ephedrae Herba, Scutellariae Radix, Acori Gramineri Rhizoma, Polygalae Radix, Typhae Pollen, Armeniacae Semen, Menthae Herba. In this study, marker substances in n-butanol fraction (SH-21-B) from Gami-Samhwang-San were analyzed by high performance liquid chromatography (HPLC) and acute toxicity of standardized SH-21-B was evaluated by good laboratory practices (GLP) guideline of Korea Food and Drug Administration. Therefore we confirmed that there were baicalin of 15.92%, amygdalin of 6.57% and ephedrine of 2.49% in SH-21-B. SH-21-B was administered in rats at dose of 0 mg/kg, 2,000 mg/kg, and 5,000mg/kg. Clinical signs of both sexes of rats were observed daily for 14 days after single oral administration. Two female rats one administered at 2,000 mg/kg and the other administered at 5,000 mg/kg, died, but no dead animal was observed among male rats. Therefore $LD_{50}$ in the female rat is observed to be 8,710 mg/kg, and MLD (Minimun Lethal Dose) of the male rat is observed to be more than 5,000 mg/kg.