The purpose of this study was to investigate the appropriate proportion of Caco-2 and HT29 co-culture in vitro cell models for permeability studies. The results showed that the transepithelial electrical resistance values of 9:1 and 1:0 groups (263?±?3.61 and 300?±?7.55) after 21-day culture were >250?Ω?cm(2), which were suitable for further experiments. The confocal laser microscopy showed that the group of 9:1 (Caco-2:HT29) had the highest integrity, whereas the group of 0:1 (Caco-2:HT29) exhibited the lowest. The staining study confirmed that mucus was successfully produced by HT29 cells, and it was also produced in co-cultures with Caco-2 cells model, but the Caco-2 monocultures did not have any blue staining, which made us affirm that mucus is only produced in the presence of HT29 cells. The real-time PCR results showed that the total highest expression level of ALPi and MUC5AC was the ratio of 9:1 (Caco-2:HT29) and lowest is 1:1 (Caco-2:HT29). So we concluded that 9:1 (Caco-2:HT29) is the optimal Caco-2 to HT29 ratio in the in vitro model co-culture for permeability studies.