CO2 removal via membrane oxygenators has become an important and reliable clinical technique. Nevertheless, oxygenators must be further optimized to increase CO2 removal performance and to reduce severe side effects. Here, in vitro tests with water can significantly reduce costs and effort during development. However, they must be able to reasonably represent the CO2 removal performance observed with blood. In this study, the deviation between the CO2 removal rate determined in vivo with porcine blood from that determined in vitro with water is quantified. The magnitude of this deviation (approx. 10%) is consistent with results reported in the literature. To better understand the remaining difference in CO2 removal rate and in order to assess the application limits of in vitro water tests, CFD simulations were conducted. They allow to quantify and investigate the influences of the differing fluid properties of blood and water on the CO2 removal rate. The CFD results indicate that the main CO2 transport resistance, the diffusional boundary layer, behaves generally differently in blood and water. Hence, studies of the CO2 boundary layer should be preferably conducted with blood. In contrast, water tests can be considered suitable for reliable determination of the total CO2 removal performance of oxygenators.