Acute myocardial infarction (AMI) involves complex molecular events, including alterations in protein phosphorylation. In this study, we performed a comparative phosphoproteomic analysis of serum samples from AMI patients and healthy controls to identify AMI-associated phosphorylation signatures. Serum samples from eight AMI patients and two pooled controls underwent high-abundance protein depletion, enzymatic digestion with Trypsin/Lys-C, and phosphopeptide enrichment using Fe-NTA spin columns. Enriched phosphopeptides were analyzed by LC-MS/MS using a Q Exactive™ HF-X Orbitrap system.