The accumulation of malignant ascites in the peritoneal cavity is a hallmark of high-grade serous ovarian cancer (HGSC). This fluid contains three-dimensional multicellular aggregates known as spheroids, which contribute to chemoresistance and are an accessible source of tumor material for proteomic-based biomarker discovery studies. Although heterogeneous ascitic spheroids can be generated from primary cell suspensions for ex vivo applications, they suffer from long generation times and reduced biological relevance. Here, we compare their ex vivo chemotherapy responses and proteomes to native spheroids that are collected directly from HGSC ascites, with the aim of assessing their suitability for proteomic-based chemoresponse prediction strategies that yield results within a clinically relevant time frame. We demonstrate that the chemoresponses of native spheroids better correlate with patients' clinical treatment responses in 4 of 5 cases and that their proteomes uniquely segregate according to ex vivo carboplatin response along the first component. This pilot study suggests key proteins and biological pathways that may facilitate a global proteomic-based screening strategy for personalized HGSC treatment, with particular emphasis on extracellular matrix proteins. As such, native spheroids have the potential to progress the personalized treatment of HGSC patients with malignant ascites.