Glycation of whey proteins leads to changes of the nutritional and functional properties of the proteins. Lactosylation of β-lactoglobulin was monitored under conditions varying with respect to temperature, water activity (a w ), and pH. The rate of the overall lactosylation and the average lactosylation degree increased with temperature (50–70 °C) and a w of 0.51–0.64, but were only slightly affected by reaction mixture pH (pH 5–7) before drying. The reaction seemed to occur in two phases, the transition between which was related to a lowering of the glass transition temperature of the system. Detailed kinetic analysis of the initial step showed that the pseudo first-order rate constants for formation of mono-lactosylated β-lactoglobulin in general were higher than the rate constants for formation of di-lactosylated β-lactoglobulin. However, the temperature dependencies of the two reaction steps were similar, corresponding to an activation energy of about 100 kJ mol −1 .