Dairy industries are interested in knowing the heat treatment undergone by milk so as to control the quality of drinking milks or to control their heating systems. Among the different techniques available to characterize the heat treatment of milk, estimation of the denaturation of proteins has been widely used. However, because the concentration of the proteins in raw milk can fluctuate significantly, determining only the concentration of a native protein without knowing its concentration in the raw milk before undergoing heat treatment can lead to significant imprecision. The objective of this study was to develop, on Biacore 3000, a biosensor assay for determining the denaturation index of R-lactalbumin by quantifying separately the native and "heat-denatured" forms of R-lactalbumin with specific monoclonal antibodies. R-Lactalbumin denaturation index is independent of the concentration of R-lactalbumin in the original raw milk. The technique developed is discriminating, fast, repeatable, fully automated, and requires no pretreatment of the milk sample.