The denaturation (loss of protein through aggregation and/or change in secondary structure) and physical characteristics such as powder morphology, particle size and size distribution, amorphous/crystalline behavior, and solubility of whey protein isolate (WPI) were investigated in a spray-drying process. The protective efficacy of sugars (lactose and trehalose) and low-molecular-weight surfactant polysorbate-80 (Tween-80) on the secondary structure (-turn, -sheet and -helix) and physical characteristics of spray-dried WPI was quantified. The WPI, WPI+sugar, and WPI+Tween-80 formulations were spray dried maintaining the total solids at 10% (w/w). The inlet and outlet temperatures were maintained at 180 and 80 degrees C, respectively. The results showed that the loss of protein through denaturation and aggregation was not significant (p>0.05). However, a significant (p<0.05) alteration of the secondary structural elements was observed. Due to spray drying of WPI without protectants, the -sheet and -turn were decreased by 4.4 and 14.5%, respectively, and the random coil increased by 20.7%. The -helix of WPI remained unaltered during the spray-drying process. The presence of Tween-80 effectively protected the -helix and -sheet but the -turn remained vulnerable and was decreased. No significant (p>0.05) change in the solubility of WPI was observed due to spray drying. Spray drying of WPI+sugar produced essentially amorphous particles. The dried powder particles were spherical with wrinkled or folded surface.