The development of personalized therapies for ovarian carcinoma patients is still hampered by several limitations, mainly the difficulty of predicting patients’ responses to chemotherapy in tumor cells isolated from peritoneal fluids. The main reason for the low predictive power of in vitro assays is related to the modification of the cancer cells’ phenotype induced by the culture conditions, which results in changes to the activation state and drug sensitivity of tumor cells compared to their in vivo properties. We have defined the optimal culture conditions to set up a prognostic test to predict high-grade serous ovarian carcinoma (HGSOC) patients’ responses to platinum chemotherapy. We evaluated the effects of hyaluronic acid (HA) and fibronectin matrices and the contribution of freezing/thawing processes to the cell response to platinum-based treatment, collecting spheroids from the ascitic fluids of 13 patients with stage II or III HGSOC. Our findings indicated that an efficient model used to generate predictive data for in vivo sensitivity to platinum is culturing fresh spheroids on HA, avoiding the use of previously frozen primary tumor cells. The establishment of this easy, reproducible and standardized testing method can significantly contribute to an improvement in therapeutic effectiveness, thus bringing the prospect of personalized therapy closer for ovarian carcinoma patients.