Introduction Volume recommendations of 80-200ml have been proposed for peritoneal fluid cytology. While cut-offs are impractical when volume is limited by the amount present and disease factors, collections, however, can be repeated. This study addresses adequacy and number needed to diagnose by comparing diagnostic agreement to volumes in single specimens, total volumes collected daily, and within admissions. The diagnostic yield of repeating collection within a single day, admission, and throughout admissions of a patient’s lifetime was also investigated. Methods Peritoneal fluid cytology specimens over a 27-year period were retrieved, and matched by collection date, admission number and patient number. Case notes were reviewed to establish all cases of malignant ascites. Results In total, 19,392 specimens from 14,327 admissions and 11,089 patients were retrieved, with 1,531 patients confirmed with malignant ascites. Agreements between cytologic diagnoses within the same day and admission were high (κ>0.8). Fluid volume increased with grade of cytologic diagnosis (p<0.001), and greater volume was associated with higher discordance (p<0.05). Specimens of 60-100ml showed the best diagnostic concordance. To achieve a 99.5% diagnostic rate, three sequential aliquots, collections from two different days in an admission, or three admissions within a lifetime is required. The diagnostic yield of one aliquot within batches from the same day was only 88.9%. Gastrointestinal (p=0.040), gynecologic (p=0.005) and lung (p<0.001) malignancies required the least repeats for diagnosis. Conclusions Omission of any fluid from laboratory submission is strongly discouraged. As a simple rule, three repeats are necessary for excluding malignant ascites.