Bioaccessibility of aflatoxin B1 (AFB1) found in contaminated peanut meal was assessed by an in vitro system that simulated conditions of Nile tilapia (Oreochromis niloticus) intestine. Peanut meal containing 17.1 µg kg−1 AFB1 was used as substrate. Reference assays were carried out with intestinal enzyme extracts from adult tilapia under realistic physiological conditions of enzyme/substrate ratio (0.21 U mg−1), pH (8.2), and temperature (25 °C). Different factors that could potentially affect bioaccessibility of AFB1 were evaluated: total digestion time (3 to 6 h), relative amount of substrate (250 to 750 mg), and amount of rice husk used as adsorbent (1:1 and 1:2, peanut meal/rice husk). AFB1 released after digestive hydrolysis was extracted by QuEChERS and quantified by a multi-reader. Bioaccessibility of AFB1 increased with digestion time (from 36 after 3 h to 100% after 6 h) and with the amount of substrate. In contrast, rice husk reduced bioaccessibility of AFB1 by 28 and 20% in ratios of 1:1 and 1:2, respectively. Therefore, this study suggests not only that in vitro assays may be used as prospective assays to evaluate bioaccessibility of AFB1 under species-specific digestive conditions, but also that inclusion of rice rusk in feed may be a promising tool to reduce mycotoxin bioaccessibility. [ABSTRACT FROM AUTHOR]