Abstract: Hyperlipidemia is a chronic disease characterized by elevated cholesterol and triglyceride levels in the blood, and has been identified as a common risk factor for many cardiovascular diseases, such as atherosclerosis, heart disease, stroke and obesity. Blood, an easily accessible non-invasive specimen that contains a variety of proteins, offers insights into pathological changes. Understanding the protein expression changes in hyperlipidemia patients after lipoprotein apheresis is important for identifying proteins related to hyperlipidemia, which is crucial for preventing or mitigating the progression of hyperlipidemia. In this study, a method of high-performance liquid chromatography-mass spectrometry (LC-MS/MS) combined with data-independent acquisition (DIA) was used to explore the effects of lipoprotein apheresis on patients with hyperlipidemia. Eight paired serum samples were included, which were collected from eight patients prior to and 2 h after apheresis. To ensure the reliability and sensitivity of proteomic measurements, the sample pretreatment protocol was optimized, with acetone precipitation selected as the optimal method for protein extraction and purification, thus effectively removing interfering substances. Under the optimized pretreatment conditions, a total of 13 up-regulated and 7 down-regulated proteins (two-tailed Student’s t-test, p<0.05 and |log₂FC|>0.585) were identified by proteomic analysis. In addition, the time-series protein expression changes in a single patient were tracked at four points (before, 2 h, 2 days, and 7 days after lipoprotein apheresis) using the same DIA LC-MS/MS approach. A total of 29 differentially expressed proteins were screened out. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were further performed on these proteins. The results showed that neutrophils associated proteins-ELANE (neutrophil elastase), MPO (myeloperoxidase), CTSG (cathepsin G), and LTF (lactotransferrin) found in proteomics before and after apheresis, as well as platelet associated proteins-PF4 (platelet factor 4), PPBP (pro-platelet basic protein) found in time-series proteomic analyses, were all associated with the neutrophil extracellular trap network (NETs) pathway. Furthermore, the expression levels of neutrophil related proteins were uniformly down-regulated, whereas those of platelet related proteins exhibited a trend of first increasing and then decreasing. In conclusion, these results suggested that lipoprotein apheresis in patients with hyperlipidemia may directly or indirectly affect the formation of NETs pathways in the body and alleviate the potential risks of inflammation and thrombosis, which provides a valuable theoretical basis for preventing or mitigating the progression of hyperlipidemia. A major limitation of this study is the relatively small sample size, therefore, additional validation with a larger cohort of clinical samples will be required in future research to confirm these findings.