In this study, we applied a quantitative proteomic approach using data-independent acquisition mass spectrometry (DIA-MS) to analyze protein expression profiles during three critical stages of anther development—pollen mother cell (Pms), tetrad (Tds), and mononuclear (Ms) stages—in the CMS line C2P5A and its maintainer line C2P5B in cotton. A total of 498 significantly differentially expressed proteins (DEPs) were identified, with 194 upregulated and 304 downregulated.