Abstract: High performance liquid chromatography (HPLC) method was studied for simultaneously determining the energy source of rat myocardial tissue, including phosphocreatine and five kinds of adenosines phosphates as adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), coenzyme I and adenosine cyclophosphate. The method can provide technique for the research on anti-cardiac insufficiency drug. The pre-cooled perchloric acid was added into the rat myocardial tissue. The homogenate of the myocardial tissue was prepared at low temperature and was centrifuged to get supernatant. After the pH value adjustment to the neutral state and centrifugation, the supernatant was stored in a -20 ℃ refrigerator.Before the HPLC determination, the supernatant was centrifuged again for 6 min, and 2 μL supernatant was used for the chromatographic analysis. With the Ultimate AQ-C18 chromatographi ccolumn, a mobile phase of methanol-phosphate buffer (10 mmol/L, pH6.2) with gradient elution and determination at 210 nm were used in the HPLC analysis. The column temperature was 15 ℃. The method is validated in terms of repeat ability, linearity and recovery rate.It has been applied in the real sample determination, presents accurate and reliable results, and is easy to operate.