Abstract: In order to investigate the potential active components and mechanism of action of Xintongshu Spray (XTS) in the treatment of acute myocardial infarction (AMI), ultra performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UPLC-Orbitrap-HRMS) was used to analyze and identify the blood-entry prototype components and metabolites of XTS in both positive and negative ion modes. A total of 41 blood-entry prototype components and metabolites were identified, including 16 phthalides, 15 organic phenolic acids, 4 monoterpenes, 3 acetophenones, 2 coumarins, and 1 sugar; 46 metabolites were identified, including 22 phthalide-related metabolites, 16 organic phenolic acids, 7 acetophenone-related metabolites, and 1 monoterpene-related metabolite. Combined with network pharmacological analysis, the target of action and signaling pathway of XTS into blood components for the treatment of AMI were predicted. The results showed that organic phenolic acids, phthalides, monoterpenes are the main active components of XTS for AMI, through the regulation of PI3K/Akt pathway, AGE-RAGE pathway, MAPK pathway to treat AMI. The results of molecular dynamics simulation showed that that the target proteins have good binding ability with the active ingredients. The use of all experimental animals has been approved by the Ethics Committee of Laboratory Animal of Anhui University of Traditional Chinese Medicine (AHUCM-rats-2021105).