Repeated phlebotomy for laboratory diagnostic testing is a known cause of iatrogenic anaemia and in critically ill neonates often leads to blood transfusion being required. This study has developed a spreadsheet clinical decision support tool to allow neonatal staff to determine the true minimum blood volume required to analyse groups of blood tests and modelled its potential benefit compared with the existing system in use. The tool calculates the minimum blood volume accounting for novel factors including the current patient haematocrit for plasma/serum samples, instrument minimum test and dead volumes (including those where shared) and sharing of samples within/between laboratory departments. A year of neonatal unit laboratory requests were examined comparing the volumes and containers of blood recommended by the hospital information system with both the amount actually collected by staff and that recommended by the tool. A total of 463 patients had 8481 blood draws for 23,899 tests or test profiles over the year. The hospital information system recommended collecting 11,222 mL of blood into 18,509 containers, while 17,734 containers were actually received (10,717 mL if fully filled). The tool recommended collecting 4915 mL of blood into 15,549 containers. This tool allows neonatal intensive care unit staff to objectively determine the minimum blood volume required for a combination of tests and is generalizable between laboratory instruments. Compared with the hospital information system, use of the minimum blood volume clinical decision support tool could maximally reduce the volume of blood collected from this neonatal unit by more than a half. Neonatal intensive care unit staff had apparently already gone some way to determining their own minimum volumes required.