In human health risk assessment, ingestion of food is considered a major route of exposure to many contaminants, although the total amount of an ingested contaminant (external dose) does not always reflect the quantity available for the body (internal dose). In this study, two in vitro methods were applied to study bioaccessibility and intestinal membrane integrity of cells exposed to patulin, a mycotoxin with significant public health risk. Seven artificially contaminated fruit juices were assayed in the presence or absence of a standard meal, showing a significant difference for bioaccessibility values between contaminated samples alone (mean 27.65 ± 13.50%) and combinations with a standard meal (mean 7.89 ± 4.03%). Different concentrations of patulin (PAT) and cysteine (CYS) (protector agent) were assayed in Caco-2 cells monolayers. At 95 μM, PAT produced a marked decrease in transepithelial electrical resistance (TEER). This effect was significantly reduced when 400 μM and 4000 μM CYS was added to the cells. Combined use of in vitro digestion models with other techniques using intestinal cell lines, such as in vitro intestinal absorption models that use Caco-2 cells, may offer a more comprehensive model of what is occurring during digestion and absorption processes. The study of beneficial effects of protective agents would also be enhanced.