Hypotension during haemodialysis and fluid overload between treatments are major problems for haemodialysis patients. Clinical means of assessing hydration state can be relatively imprecise. We describe a non-invasive method of measuring absolute blood volume (BV) during a mock in vitro haemodialysis session which adds objective information to that assessment. As fluid is removed by ultrafiltration, haemoglobin concentration [Hb] rises proportionately with the fall in BV. An optical monitor clamped across the transparent dialysis tubing gives a continuous readout of near infra-red light transmitted through the blood, and this can be converted to [Hb] values. The net change in BV is the difference between the volume of fluid ultrafiltered and the volume which refills the vascular compartment from the extravascular space. By analysing the change in [Hb] and therefore the change in BV at two different rates of fluid removal, the absolute BV can be determined. The accuracy of this method was tested in vitro. This optical method accurately measures the change in BV over a range of [Hb] from 4 to 15 g/dl and blood circulation pump speeds of 150-300 ml/min. A series of 10 in vitro experiments was performed. The mean relative difference between the measured BV and the calculated BV, was 5.7 +/- 2.5%. This readily repeatable technique can accurately measure BV during a mock in vitro haemodialysis session, thus providing information for the clinical assessment of the hydration state. Information from these experiments will assist in future in vivo studies.