In the evaluation of the oral bioavailability of a mycotoxin, the first step is the determination of its bioaccessibility, i.e. the percentage of mycotoxin released from the food matrix during digestion in the gastrointestinal (GI) tract that could be absorbed through the intestinal epithelium. Different in vitro digestion models have been recently used for determination of bioaccessibility, thereby avoiding the use of more complex cell culture techniques or the use of animals in expensive in vivo experiments. In vitro methods offer an appealing alternative to human and animal studies. They usually are rapid, simple and reasonably low in cost, and can be used to perform simplified experiments under uniform and well-controlled conditions, providing insights not achievable in whole animal studies. The available in vitro methods for GI simulation differ in the design of the system, the composition of the physiological juices assayed, as well as in the use or not of intestinal microbiota. There are models that only simulate the upper part of the GI tract (mouth-stomach-small intestine), whereas other methods include the large intestine, so that the model chosen could have some influence on the bioaccessibility data obtained. Bioaccessibility depends on the food matrix, as well as on the contamination level and the way the food/feed is contaminated (spiked or naturally). This review focuses on the currently available data regarding in vitro digestion models for the study of the bioaccessibility or absorption of mycotoxins, detailing the characteristics of each digestion step and the importance of the physiological juices employed during digestion. The effect that different factors play on mycotoxin release from the food matrix in the GI tract is also considered, and existing data on bioaccessibility of the main mycotoxins are given.