The ovarian tissues of 3 mice aged 3 months in the control group and the hyperhomocysteinemia group were selected for advanced 4D Laber-free quantitative proteomic analysis. According to 4D Laber-free quantitative proteomics analysis, we successfully quantified 56,611 peptides and identified 5,764 quantifiable proteins. Principal component analysis (PCA) highlights the extraordinary quantitative repeatability between biological replicas. Subsequently, we screened for differentially expressed proteins (DEPs) (P <0.05, multiple change (FC) >1.5). The results showed that there were 317 up-regulated and 113 down-regulated proteins.