The present work developed a non-affinity-based chromatographic method to enrich MUC16 from serum. The enriched MUC16 sample was further processed using a Midi Top 14 abundant protein depletion column. Peptides identified using bottom-up proteomics yielded 1–8% coverage of MUC16. Additionally, MUC16 was detected in samples containing less than the clinical cut-off level of CA125 (35 U/mL), suggesting that this strategy of enrichment and bottom-up proteomics can enable analysis of CA125 from serum of individuals with early-stage ovarian cancer and those whose tumors express CA125 (MUC16) at low levels.