The invention relates to a substance detection method, specifically a method for quantitatively detecting ATP, ADP, and AMP in tissue. According to precedence order, the method includes preparing reagent-preparing solution-setting chromatographic condition-making standard curve-preparing solution of sample to be detected-measuring content of specimen. The method is suitable for wide range, applicable for quantitative measurement of ATP, ADP, and AMP in the fields of clinical examination, scientific research, medicolegal expertise, etc.; the method can detect the content of ATP, ADP, and AMP in tissue with high efficiency, has the advantages of high sensitivity, high stability, high repeatability, high precision, and high recovery rate; the method can separate ATP, ADP, and AMP in tissue within 10 minutes, effectively reduce the interference of impurities; the reagent and chemicals used in the detection method are cheap, can be easily prepared, and have low cost, the invention has rapid detection and short time, facilitates to detect in batch, and is simple for operation.