(1) Background: More than 35% of women with ovarian cancer develop ascites, a build-up of fluid in the peritoneal cavity caused by increased vascular permeability and/or obstructed lymphatic drainage. Malignant ascites contains three-dimensional tumor cell clusters called spheroids, stromal cells, cancer-associated fibroblasts, and blood cells. Although improved cancer cell viability in ascites is well-described, the molecular changes in response to ascites remain poorly understood. (2) Methods: We performed a comparative proteomics analysis of patient’s ascites compared to serum; and of cancer cells grown adherent or as spheroids in ascites compared to tissue culture medium. The impact of ascites or supplementing tissue culture medium to the phenotype, viability and paclitaxel response were investigated. (3) Results: The ascites proteome showed an increase of extracellular proteins and secreted proteins compared to serum. Growing ovarian cancer cells in ascites altered viability and morphology, correlating with increased abundance of extracellular matrix-receptor interaction proteins. However, this did not significantly impact their response to chemotherapy treatment. Addition of chemokines or periostin to cell culture medium was unable to mimic the phenotype observed when cells were grown in as-cites (4) Conclusions: Ascites enhances ovarian cancer cell proliferation and drives molecular changes, emphasizing its importance in modelling disease and regulating extracellular matrix deposition.