Residual endotoxins, commonly associated with bacterial biofilms colonizing reusable medical devices have been associated with pyrogenic reactions in patients. We have used a quantitative, sensitive and reproducible kinetic chromogenic adaptation of the Limulus Amebocyte Lysate assay to assess endotoxin recovery from an in-vitro bacterial biofilm. The 'recovery method' was based on a combination of physical treatment (vortexing and sonication) and chemical treatment (immersion in recovery solution). Five recovery solutions were investigated. The recovered endotoxin was greater when the biofilm was treated with a 1% SDS solution. The sensitive and reproducible method we have developed should allow the recovery and measurement of biofilm bacterial endotoxins on implanted and colonized medical devices. Moreover, the amount of endotoxin was sufficient (> 1000 endotoxin units/cm2 of substrate) to enable a substantial reduction by sterilization processes, the efficiency of which on biofilm endotoxins has yet to be proven.