BACKGROUND The study aims to investigate the limitation of a poultry digestive tract model developed by Menezes‐Blackburn et al. J Agric Food Chem 63: 6142–6149 (2015) on the evaluation of the bioefficacy of phytases. RESULTS It was confirmed that the in vitro model does not mimic the in vivo situation in the birds sufficiently well to identify the best phytase product under real conditions, or to draw conclusion on the effect of phytate concentration, phytate source or feed composition on the bioefficacy of phytase. Addition of calcium ion (Ca2+) up to a concentration of 10 g kg−1 to the feed substrate, for example, did not affect enzymatic phytate dephosphorylation in the in vitro model in contrast to the observation in poultry. CONCLUSION The in vitro approach was shown to be applicable as a complementary tool in the pre‐selection of promising phytase candidates, resulting in a reduction in the number of feeding trials in the initial screening phase. © 2020 The Author. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.