The patient was a 51-year-old Japanese female who had been diagnosed with hyperlipidemia. At the first medical examination, her serum levels of total cholesterol (TC) and triglyceride (TG) were 482 and 205 mg/dl, respectively. Since hyperlipidemia was not improved by pravastatin, atorvastatin or niceritrol, and since the levels of thyroid-stimulating hormone (TSH) and free T4 were 730 IU/ml and 0.3 ng/dl, respectively, the patient was diagnosed as secondary hyperlipidemia with hypothyroidism. A method for the charge isolation of lipoproteins using capillary isotachophoresis (cITP) is proposed as a clinical application because it allows us to quantitatively measure electronegative low-density lipoprotein cholesterol (LDL-C), a potent marker of coronary heart disease. After 5 months of treatment with levothyroxine, Serum TC and LDL-C levels drastically decreased without statin treatment and high-density lipoprotein cholesterol (HDL-C) increased. In the lipoprotein profiles as assessed by cITP after treatment with levothyroxin, all HDL-C subfractions were increased and fast-migrating LDL/electronegative LDL appeared to be greatly reduced after treatment, while the area under the non-modified LDL peak was increased. The cITP analysis was able to obtain more information about coronary risk factors and may be clinically useful for evaluating the effect of treatment with levothyroxine in patients with hypothyroidism and secondary hyperlipidemia.