The removal of middle molecules during continuous extracorporeal detoxification therapies is a scientific field of increasing attention. However, it is difficult to find suitable and inexpensive markers and methods regarding the in vitro clearance of molecules in a weight range between 10 and 20 kDa. We present an easy and reliable cytochrome C-based photometric method to compare the in vitro clearance of different hemofilters during hemodialysis therapy. Cytochrome C is an inexpensive globular protein that shows a relative absorption maximum at λ = 410 nm, allowing photometric concentration assessment in the dialysate outflow line. Various hemofilters were evaluated to assess the removal of cytochrome C during simulated continuous venovenous hemodialysis. Although reported sieving coefficients for globular proteins and surface area of all hemofilters were similar, clearance differences of up to 55% were observable at blood and dialysate flows of 150 ml/min and 4,000 ml/h, respectively. We found that even in a strict hemodialysis setting with moderate blood flow, a remarkable diffusive cytochrome C clearance can be achieved. Hereby, diffusion is eventually supported by transmembrane flow and backflow in the proximal and distal sections of the hollow fiber of a hemofilter.