The molecular mechanisms associated with spaceflight-induced biological adaptations that may affect many healthy tissue functions remain poorly understood. In this study, we analyzed temporal changes in the serum proteome of six astronauts during prolonged spaceflight missions using quantitative comprehensive proteome analysis performed with the data-independent acquisition method of mass spectrometry (DIA-MS). DIA-MS successfully identified 624 non-redundant proteins in sera and further quantitative analysis for each sampling point provided information on serum protein profiles closely related to several time points before (Pre-), during (In-), and after (Post-) spaceflight.