A sensitive and specific HPLC-UV method has been developed, for the first time, to simultaneously quantify 12 bioactive ingredients in Ligusticum chuanxiong Hort. (Rhizoma Chuanxiong). This assay was fully validated in respect to precision, accuracy and sensitivity. This method was successfully applied to quantify twelve ingredients in six different Chuanxiong samples. The results demonstrated significant variations in the total content and quantity of each of the main bioactive compounds in different herbs, indicating that quality control of bioactive ingredients in Chuanxiong is critical to ensure its clinical benefits. This assay can be readily utilized as quality control method for Chuanxiong.