The present invention has been made in order to solve the problems in conventional methods of assaying an endotoxin in a specimen such as plasma or serum by the limulus test, which requires a complicated pretreatment procedure such as centrifugation for removing denatured precipitates formed with an acid treatment. According to the assay method of the present invention, an endotoxin adsorbed by proteins, lipids and platelets can be efficiently liberated simply by adding a mixed aqueous solution having a specific composition according to the present invention without any separation procedure and thus a sample solution of good qualities can be prepared. After adding the mixed aqueous solution containing a specific surfactant, a compound having an imidazolyl group or an amino group and an alkaline earth metal salt and an alkaline metal hydroxide to the specimen, followed by addition of the limulus amoebocyte lysate components, the endotoxin can be easily, quickly and precisely assayed. In particular, gram-negative bacterial septicemia, which can be hardly diagnosed, can be quickly assayed thereby.