Background: DIAAS values are based on true ileal digestibility of amino acids, performed in human or pig trials and are internationally recognized as the Gold standard. However, in vivo experiments are expensive and have ethical concerns. Therefore, the FAO recommends the establishment and validation of in vitro systems as screening tools for digestibility assessments. Methods: The static INFOGEST in vitro digestion system is a robust and reproducible tool, closely mimicking physiological digestion. Towards the goal of establishing an in vitro digestibility protocol, intestinal digesta were separated in undigestible and digestible fractions by precipitation and both fractions were analyzed for total nitrogen (Kjeldahl), or after acidic hydrolysis analyzed for total amino groups (OPA) and individual amino acids (HPLC), respectively. Results: From TAA results, digestibilities of individual amino acids and in vitro DIAAS were calculated. Physiological relevance of the in vitro results was demonstrated by comparison with in vivo values from human (WPI, zein), pig (WPI, zein, collagen, black beans, pigeon peas, all-bran) and rats (peanut). In vitro digestibility and in vitro DIAAS were in good agreement with in vivo values. Conclusion: In conclusion, the newly developed in vitro digestibility protocol could be a complementary tool for screening and comparison of different protein sources.