Accurate enumeration of specific Bacillus strains in probiotic formulations is challenging for both research and industry. Conventional plate counts cannot reliably distinguish strains, causing inconsistencies in viability assessment and potentially affecting product quality and applications. The present study addresses this limitation by developing a viable count method tailored for the selective enumeration of Bacillus spores in a blend of three proprietary strains: Bacillus coagulans LBSC, Bacillus subtilis PLSSC, and Bacillus clausii 088AE based on optimized pH and antibiotic selection pressures. Spore counts on standard and selective media correlated strongly for LBSC (P=0.4736), PLSSC (P=0.3387), 088AE (P=0.4721). Colony morphology, microscopy, biochemical testing and 16S rRNA sequencing eliminated potential ambiguities. The method met the validation criteria of specificity, precision, intermediate precision, linearity and accuracy for working range of blends with concentrations 0.36 to 36×109 CFU/g (0.12 to 12×109 CFU/g of each Bacillus strain in the blend). Specificity showed no growth of non-target bacteria. Precision and intermediate precision) was < 10 % RSD and a statistically significant linear regression model confirmed linearity (R2>0.99, F>1000, p<0.001). Accuracy was demonstrated with no significant difference in the means of theoretical and observed spore count (P-value>0.05). This innovative approach provides a simple guiding solution to the limitations of current enumeration techniques and a valuable contribution to probiotic research for enhancing the application of Bacillus-based probiotics.