HiPSC-derived blood–brain barrier (BBB) models are valuable for pharmacological and physiological studies, yet their translational potential is limited due to insufficient cell phenotypes and the neglection of the complex environment of the BBB. This study evaluates the plasma compatibility with hiPSC-derived microvascular endothelial-like cells to enhance the translational potential of in vitro BBB models. Therefore, plasma samples (sodium/lithium heparin, citrate, EDTA) and serum from healthy donors were tested on hiPSC-derived microvascular endothelial-like cells at concentrations of 100%, 75%, and 50%. After 24 h, cell viability parameters were assessed. The impact of heparin-anticoagulated plasmas was further evaluated regarding barrier function and endothelial phenotype of differentiated endothelial-like cells. Finally, sodium-heparin plasma was tested in an isogenic triple-culture BBB model with continuous TEER measurements for 72 h. Only the application of heparin-anticoagulated plasmas did not significantly alter viability parameters compared to medium. Furthermore, heparin plasmas improved barrier function without increasing cell density and induced a von Willebrand factor signal. Finally, continuous TEER measurements of the triple-culture model confirmed the positive impact of sodium-heparin plasma on barrier function. Consequently, heparin-anticoagulated plasmas were proven to be compatible with hiPSC-derived microvascular endothelial-like cells. Thereby, the translational potential of BBB models can be substantially improved in the future.