The aim of this study was to develop whey protein-pullulan (WPI-pullulan) microcapsules in order to assess its protective effect on the viability probiotic bacteria under in vitro gastrointestinal conditions. The probiotic bacteria used in this study was of Lactobacillus acidophilus NRRL-B 4495. This study demonstrated that probiotic bacterial cells were successfully encapsulated into WPI-pullulan gel matrix. The appropriate microcapsule formation parameters include formation of primary emulsion having 50% inner polymer aqueous phase having whey protein in the presence of 10.8% pullulan with sunflower oil. Optimal concentration of bacteria for microencapsulation was decided to be kept at 10.00 log CFU/g since increasing initial load resulted in decreasing microencapsulation efficiency. The incorporation of pullulan into whey protein wall matrix created spherical microcapsules with denser and smoother surface structure. Microcapsules with varying pullulan concentrations in the wall matrix obtained the mean diameters between 68 μm to 80μm. The survival rates of Lactobacillus acidophilus NRRL-B 4495 under in vitro gastrointestinal conditions were significantly (p<0.05) improved by the presence of pullulan. WPI-pullulan wall matrix resulted in a 87.18% survival rate as compared to 73.19 % for free cells after 3h incubation in simulated gastric solution. Protein-polysaccharide gel network formed in the microcapsules restricted diffusion of bile salts, as well. During spray drying experiments with high concentration of pullulan, fibrous structure formation was observed. Optimal conditions for spray drying of probiotic cells were decided to have 145 ºC as an outlet temperature in the presence of WPI-pullulan wall matrix with 4.5:1 protein:pullulan ratio. Moreover pullulan incorporation in contributed to release properties of microcapsules by altering the dissolution and porosity properties. On the other hand, encapsulation into developed polymer wall matrix in wet and spray dried forms did not provide any positive advantage for the responses of probiotic microorganism against heat stress.