The heat denaturation of whey proteins affects the functional properties of milk. Correlations of β-lactoglobulin (β-LG) denaturation to gelation time, gel firmness, and gel moisture content have been widely documented. Currently, no technique is available for quantifying β-LG denaturation in milk without altering its native composition or requiring a laborious procedure. The goal of this study was to establish if an optical backscatter response of whey protein denaturation during milk heat treatment could be determined that would be the basis for an inline optical measurement technology. The experimental design consisted of 1 factor (time at 80°C) and 6 levels (0, 3, 5, 7, 12, and 25 min). Physicochemical analysis performed indicated that β-LG denaturation followed a first-order response during thermal treatment. The light backscatter response, measured as a ratio of two 25-nm wave bands (832.5 nm/382.5 nm), significantly correlated to β-LG denaturation and had a 14% increase for milk with 75% β-LG denaturation. The strength of the optical response at the proposed wave bands and their correlation to denaturation suggests that light backscatter could potentially be used to measure β-LG and other whey protein denaturation inline.