Background: The REZOLVE clinical trial investigated the effect of administering bevacizumab via the intraperitoneal route to reduce re-accumulation of ascites in patients with ovarian cancer who were not suitable for further chemotherapy. Plasma and ascites were collected throughout for translational research. Ascites contains cell-free DNA (cfDNA), a large proportion of which is tumour derived (ctDNA). There is increasing interest in cfDNA in plasma, yet little is known of it in ascites. Objectives: To compare cfDNA in ascites and plasma in terms of total concentration, tumour proportion and endothelial-cell derived (ec-cfDNA) proportion and investigate their association with clinical outcomes (paracentesis-free interval, overall survival) and CA125 level. Methods: Longitudinal plasma and ascites samples were collected from 20/24 participants and stored at -70°C for up to 8.5 years. cfDNA was extracted from 0.3-1 mL fluid using conventional protocols. Standard and methylation-specific PCR was used to measure total cfDNA, ctDNA and ec-cfDNA. Values were correlated with time to paracentesis pre- and post-bevacizumab treatment (the primary trial outcome) as well as overall survival, using log-rank tests. Relationships with clinical CA125 levels were tested by Pearson’s correlation coefficient. Comparisons between plasma and ascites used non-parametric analyses. Results: cfDNA was detected in all samples, with higher yield in ascites (average 669 ng/mL) than plasma (average 75 ng/mL, p<0.0001). ctDNA was detected in 30/32 (94%) ascites samples and 37/56 (68%) plasma samples. ctDNA was detected in plasma and/or ascites from each patient at at least 1 time point. ctDNA proportion was higher in ascites than plasma (p<0.0001) and ec-cfDNA proportion was higher in plasma than ascites (p=0.002). High ctDNA (>75%) in ascites at baseline was associated with significantly shorter paracentesis-free interval (median interval 47.5 versus 84 days, hazard ratio (HR) 2.21, 95% confidence interval (CI) 0.85 to 5.73, p=0.039). ctDNA presence in plasma at baseline was unfavourable for survival (median survival 56 versus 242 days, HR 3.21, 95% CI 1.15 to 9.00, p=0.008). A significant positive correlation was observed between ctDNA proportion in plasma and CA125 level measured within 7 days (p=0.0006). No difference in total cfDNA, ctDNA nor ec-cfDNA was observed between participants who were bevacizumab responders and non-responders in the trial. Conclusions: Sufficient cfDNA was obtained from plasma and ascites to perform qPCR for three biomarkers. Ascites was found to contain proportionately more ctDNA, while plasma contained more ec-cfDNA. The early evidence presented here supports the potential value of cfDNA biomarkers in plasma and ascites, however incorporation of their collection in future clinical trials will allow further investigation.