Nanotechnology is finding increasing application in biology and medicine. As with other pharmaceutical formulations and medical devices intended for use in animals and human patients, contamination of nanoparticles with bacterial endotoxins should be thoroughly investigated before preclinical in vitro and in vivo characterization. Traditional methods to study endotoxin contamination include the in vitro quantitative Limulus Amebocyte Lysate test and the in vivo qualitative rabbit pyrogen test. Both of these tests have a long history of use for traditional pharmaceuticals and medical devices and are routinely used in drug development. Here we report that nanoparticles often interfere with these traditional endotoxin detection tests and suggest approaches to detect and overcome such interferences.