The bioaccessibility of pesticide residues is a crucial parameter for the prediction of risks to human health and potential exposure to pesticides. Due to various limitations of in vivo studies, in vitro digestion is imposed as a rapid and cost-effective alternative for assessing the bioaccessibility of pesticides in fruits. In this chapter, a protocol for the assessment of the bioaccessibility of pesticides in fruit is proposed and described in detail, including the collection/preparation of fruit samples, the in vitro digestion protocol, the bioaccessibility assessment, and the health risk assessment. It is a standardized in vitro digestion protocol designed to ensure better comparability of results between studies and a better understanding of the bioaccessibility of different pesticides during digestion.