Conventional measurements of kinetic constants currently in use are performed at equilibrium and may require large volumes, especially at a low association rate constant kon. If the measurements are made out of equilibrium, the values obtained may be biased by dilution of the sample with the flow of the running buffer. In some applications, the available sample volume can be very critical and requires the development of tools to measure kinetic constants with low volumes. In this paper, by combining an experimental, numerical and modeling approach, we propose a surface plasmon resonance-based method that relies on an out-of-equilibrium measurement using the effect of dilution by flow to its advantage. This new method should have a significant impact in biochemistry and medical research.