In regulatory guidelines for bioequivalence (BE) assessment, the definitions of AUC for primary assessment are different in ICH countries, i.e., AUC from zero to the last sampling point (AUCall) in Japan, AUC from zero to infinity (AUCinf) or AUC from zero to the last measurable point (AUClast) in the US, and AUClast in the EU. To assure sufficient accuracy of truncated AUC for BE assessment, the ratio of truncated AUC (AUCall or AUClast) to AUCinf should be more than 80% both in Japanese and EU guidelines. We investigated how the difference in the definition of truncated AUC affects BE assessment of sustained release (SR) formulation. Our simulation result demonstrated that AUCall/AUCinf could be ≥80% despite AUClast/AUCinf being <80% and AUCall failed to detect formulation difference. In Japanese package inserts of generic drugs in SR formulation, there were products for which AUCall/AUCinf was ≥80% though AUClast/AUCinf was <80%. In conclusion, it was confirmed that the difference in definition of truncated AUC affected the judgment of validity of truncated AUC for BE assessment, and AUCall could fail to detect the substantially different in vivo dissolution profile of generic drugs with SR formulation from the original drug.