Internal filtration in high-flux (HF) dialyzers significantly contributes to convective solute removal of molecules with poor diffusibility, but it is difficult to quantify. The aim of this study was to present the theory and to develop a method for measuring internal filtration and backfiltration in HF dialyzers, which also could be applied to patient studies. In a series of lab-bench experiments, the mean transit times (τd) of indocyanine green (ICG) passing the dialyzer were optically measured under different operating conditions and compared with mean transit times calculated from the known volume of the blood compartment (τV) using a mathematical model. τd was always larger than τV. The relative difference in mean transit times (1 - τV/τd) was related to the average cumulative filtration rate (Qfil). The internal filtration fraction Fb = Qfil/Qb was largely independent of blood flow (Qb) and not different from theoretical predictions obtained from a mathematical model. The dispersion of a nondiffusible indicator such as ICG can be used to quantify the magnitude of internal filtration and backfiltration in HF dialyzers using available technology. This information could be useful for testing the HF dialyzers in everyday situations.