Background Non-invasive measurement of solute concentrations in a binary macromolecule system is a powerful tool for the study of mass transport phenomena, particularly for studying solute-solute interactions. Our objective is to extend an existing differential refractometric technique for measurement of concentrations in a two-macromolecule system. Methods We used a specially designed cell and associated optical equipment to measure the local refractive index and light extension coefficient of a polymer mixture, The macromolecules used were dextran (molecular weight 2 million) and the protein cytochrome-c. A three-layered calibration procedure quantified the refraction and light extinction due to each of these components in a mixture, as well as the interaction between the two components. Results Calibration data showed that dextran and cytochrome-c concentrations could be determined from mixture properties with good accuracy and repeatability. Specifically, for cytochrome-c over the concentration range from 0.1-0.6mass%, the maximum uncertainty in measured concentration was approximately +/-0.013%, expressed as a 95% confidence interval for the measurement. The corresponding maximum uncertainty for dextran over the concentration range from 0.1-0.6 mass% was +/-0.037%. Conclusion The experimental approach proposed herein is suitable for the measurement of transport in binary polymer mixtures where one or both components are fairly strongly light-absorbing. It allows real-time monitoring of macromolecular transport without radioactive tracers or other complex equipment.