The theory of a method for determining active concentration of nonpurified protein samples using the BIAcore biosensor technology has been developed. The method relies on change in binding rate with varying flow rate at high ligand concentration where mass transport from bulk to surface with immobilized ligand becomes partially rate limiting. Prior study of binding kinetics is not necessary. If the molecular weight and the diffusion coefficient of the analyte protein are known, no standard with a known concentration is required. From numeric computer simulations a simple analytical expression for the mass transport coefficient derived at totally mass transport limiting conditions is shown to be applicable for conditions of partial mass transport limitation. Simple one to one association is assumed in deriving the method, but it is applicable even with more complex kinetics due to a bivalent analyte or heterogeneity of analyte or ligand.