In animal nutrition, probiotics are considered as desirable alternatives to antibiotic growth promoters. The beneficial effects of probiotics primarily depend on their viability in feed, which demands technical optimization of biomass production, since processing and storage capacities are often strain-specific. In this study, we optimized the production parameters for two broiler-derived probiotic lactobacilli (L. salivarius and L. agilis). Carbohydrate utilization of both strains was determined and preferred substrates that boosted biomass production in lab-scale fermentations were selected. The strains showed good aerobic tolerance, which resulted in easier scale-up production. For the freeze-drying process, the response surface methodology was applied to optimize the composition of cryoprotective media. A quadratic polynomial model was built to study three protective factors (skim milk, sucrose, and trehalose) and to predict the optimal working conditions for maximum viability. The optimal combination of protectants was 0.14g/mL skim milk/ 0.08 g/mL sucrose/ 0.09 g/mL trehalose (L. salivarius) and 0.15g/mL skim milk/ 0.08 g/mL sucrose/ 0.07 g/mL (L. agilis), respectively. Furthermore, the in-feed stabilities of the probiotic strains were evaluated under different conditions. Our results indicate that the chosen protectants exerted an extensive protection on strains during the storage. Although only storage of the strains at 4 °C retained the maximum stability of both Lactobacillus strains, the employed protectant matrix showed promising results at room temperature.