Assessment of the binding of pharmaceutical agents to red blood cells is important in understanding the pharmacokinetics of a compound. Pharmacokinetic parameters are usually determined by analysis of drug concentrations in plasma rather than whole blood, which may be misleading if the compound of interest partitions into red blood cells or is excluded from them. Additionally, fraction unbound values determined in plasma may not be correlated with fraction unbound in whole blood for compounds which bind to erythrocytes. This could lead to over-prediction of whole blood potency and misinterpretation of in-vivo pharmacokinetic data if not taken into account. Since some compounds show specific binding to blood cells selection of the most appropriate concentration to measure blood partitioning is crucial. If too high a concentration is selected then binding sites in the blood become saturated and partitioning is not effectively observed; too low and detection issues start to become apparent and the physiologically relevant range is missed. We have developed robust methods for assessment of both blood to plasma ratio and fraction unbound in blood, presented herein. It was found that measurement of blood to plasma ratio was more reliable using methodology where both blood and plasma samples were analysed. This is preferable to indirect methods involving analysis only of the plasma fraction, which were found to perform poorly for compounds exhibiting low blood to plasma ratios. Concentration dependency studies revealed that a concentration of 500nM was most appropriate for the measurement of blood to plasma ratio. Above 2000nM, it was found that the ratio was diminished for certain compounds to an extent that it could be missed in screening. A method for measuring fraction unbound in blood was developed using equilibrium dialysis and it was found that this was well correlated with fraction unbound in plasma, except in the case of known red blood cell binders where the measured blood plasma ratio could be used to improve the correlation. A strategy for utilising these methods in drug discovery is suggested and the use of such data in a drug discovery project exemplified. It is concluded that measurement of blood-plasma ratio at the appropriate concentration at an early stage of drug discovery leads to an early understanding of potential issues, and enables effective decisions to be made on follow up screening strategy.