Endotoxin exists on the outer membrane of Gram-negative bacteria and poses risks to human health by triggering a series of immune responses. However, the widely used Limulus amoebocyte lysate (LAL) test for endotoxin detection can be compromised by low endotoxin recovery (LER) – the failure to recover known amounts of purified lipopolysaccharide from biological formulations. Yuan Cao, Yujie Zhang & Frank Qiu summarise the possible reasons for LER from three perspectives: endotoxin standards used in hold time studies, excipients and protein active pharmaceutical ingredients. Potential mechanisms and strategies to mitigate the LER phenomenon are also discussed. (DOI: 10.1002/bip.23470)