Background Prevention of iatrogenic blood loss is an essential component of patient blood management (PBM) in intensive care units (ICUs). The amount of iatrogenic blood loss from diagnostic phlebotomy in the ICUs at Universitas Academic Hospital, Free State Province, South Africa, is unknown. Objectives To quantify diagnostic phlebotomy volumes, and volumes submitted in excess for diagnostic testing in the ICU. Methods We conducted a prospective descriptive observational study on adults who were admitted to ICUs at a single centre over a period of 14 days. The weight of each filled phlebotomy tube was calculated using the specific gravity of blood and averages of empty phlebotomy tubes, establishing the total volume. ResultsData from 59 participants with a median length of stay at the ICU of 3 days were analysed. The median phlebotomy volume was 7.0 mL day and 13.6 mL/ICU admission. The volume of blood required for analysis daily and ICU admission was 0.7 mL and 2.2 mL, respectively. The median phlebotomy volume in excess of the amount required for analysis daily and ICU admission was 5.05 mL and 12.11 mL, respectively. Conclusion While the median excess daily phlebotomy volume in this present study may seem insignificant and underestimating the true excess of phlebotomy volume, interventions to reduce phlebotomy volumes and development of a PBM guideline for appropriate phlebotomy volumes and preventing wastage of patients’ blood in the ICU is required. Contributions of the study We determined blood volume requirements for laboratory instrumentation, which allows phlebotomists to be cognisant of the true requirements for diagnostic tests to be undertaken accurately. We established diagnostic blood loss volumes in critical care units at a tertiary hospital in South Africa and we advocate for the introduction of patient blood management practice guidelines at local institutions.