The blood-to-plasma ratio (R b ) is an important property of drug candidates. R b is applied widely in drug discovery to convert plasma pharmacokinetic parameters to the respective parameters in blood and to develop in vitro-in vivo correlations. Some compounds such as prodrugs, soft drugs, and peptide mimetics are unstable in blood, making accurate in vitro R b measurement challenging, but necessary. Low temperature often reduces the rate of enzymatic and chemical reactions and increases the stability of labile compounds in biomatrices. In this study, the effects of 4°C on R b measurement were evaluated using a set of structurally diverse compounds with various binding and red blood cell (RBC) transport mechanisms. The results indicate that a 4°C R b method provides comparable R b values to the 37°C method for most compounds and can therefore be applied to measure the R b of unstable compounds in drug discovery. In some rare cases, when compounds have a high affinity to specific RBC components (e.g., carbonic anhydrase), the 4°C method may underestimate R b. In these specific cases, the use of appropriate inhibitors to stabilize unstable compounds is recommended.