Significance statementWe identified that PLGA‐PEG‐PLGA hydrogels, which have been used in human clinical trials and possess a demonstrable safety profile, induced significant cytotoxicity in conventional in vitro assays. This major contradiction may lead to inconsistent and misleading toxicology due to the limited biological representation of these assays. Cytotoxicity evaluation is a crucial element of screening the biological response to new biomaterials. However, as standard test methods do not recapitulate the in vivo environment, tailored adaptations may be required to reflect the true biological response elicited toward novel biomaterials.