Carotenoids are liposoluble pigments responsible for the colors yellow, orange, and red, which have high antioxidant capacity and pro-vitamin A activity. They are found in vegetables, fruits, algae, and some animals and can be produced by fungi and bacteria. To exert their bioactivity in humans, the carotenoids must be bioaccessible, which means they must be released from the food matrix during digestion, transferred to mixed micelles (since they are lipophilic compounds), and then absorbed by the gastrointestinal tract. In this chapter, we describe a protocol for the in vitro digestion of fruits and derived products that allows for the calculation of bioaccessibility and an optional protocol for an in vitro static fermentation batch to simulate colonic fermentation.