The consumption of probiotic products, which is proven to provide health benefits, has increased significantly in the last few years. A proper selection of strains and food matrix should be conducted for the processing of probiotic food products, because certain components in the food matrix may interact with these probiotics, altering their functional performance. Therefore, the application of probiotic strains in different food matrices could represent a great challenge to maximize their effectiveness. This research aimed to evaluate the viability of potentially probiotic strains in fermented milk prepared using different matrices. L. casei SJRP38 and L. fermentum SJRP43, previously selected by their good technological features, safety and high probiotic potential, were evaluated and used in coculture with the commercial strain of S. thermophilus TA040 for fermentation. The influence of matrices: M1 - reconstituted skim milk powder (RSMP) + 7% sucrose and M2 - RSMP + 7% sucrose + 5% flaxseed (Linum usitatissimum L.) were evaluated on the acidifying kinetic parameters and viability of the strains under simulated gastrointestinal (GI) conditions during refrigerated storage. Times to reach the maximum acidification rate, pH 5.0, and pH 4.6 (end of fermentation) were influenced by the food matrix. M2 had a negative effect on the fermentation time, causing an increase of up to 3 h for finishing the process. All strains in the M1 matrix survived well (>7 log cfu/mL) during the simulated GI, which is equivalent to the passage of bacteria through the human GI tract. M2 affected the counts of L. casei SJRP38, L. fermentum SJRP43, and S. thermophilus TA040, compared with the M1 matrix, and they had a reduction of up to 3 log cfu/mL after intestinal passage. Additionally, the population reduction after the assay was influenced by the storage period for both matrices. Considering the overall results, Lactobacillus casei SJRP38 in coculture with S. thermophilus TA040 in the M1 matrix presented a high probiotic potential for further application in functional fermented products.