Developing a mass spectrometry-based assay for the ovarian cancer biomarker CA125 (MUC16) is a desirable goal, because it may enable detection of molecular regions that are not recognized by antibodies and are therefore analytically silent in the current immunoassay. Additionally, the ability to characterize the CA125 proteoforms expressed by individuals may offer clinical insight. Enrichment of CA125 from malignant ascites may provide a high-quality source of this important ovarian cancer biomarker, but a reliable strategy for such enrichment is currently lacking. Beginning with crude ascites isolated from three individual patients with high grade serous ovarian cancer, we enriched for MUC16 using filtration, ion exchange, and size exclusion chromatography and then performed bottom-up proteomics on the isolated proteins. This approach of enrichment and analysis reveals that the peptides detected via mass spectrometry map to the SEA domain and C-loop regions within the tandem repeat domains of CA125 and that peptide abundance correlates with clinical CA125 counts.