A process for determining a bacterial endotoxin, which comprises contacting an assay sample with (A) a material selected from the group consisting of an amoebocyte lysate of horseshoe crab an a pro-clotting enzyme separated from the lysate, and (B) a peptide-type substrate of the formula (R.sub.1 --Gly--Arg--R.sub.2), wherein R.sub.1 represents a member selected from the group consisting of an L-amino acid moiety whose N-terminal is protected by a protective group, a peptide moiety consisting of an L-amino acid and protected by a protective group at its N-terminal, a D-amino acid substituted L-amino acid moiety, and a D-amino acid substituted peptide moiety consisting of an L-amino acid, and is bonded to the amino group of the glycine moiety expressed by Gly through a peptide bond; and R.sub.2 represents a moiety which is bonded to the C-terminal of an L-arginine moiety expressed by Arg through an acid amide bond and/or ester bond and can be enzymatically hydrolyzed in the presence of the material (A) and the endotoxin to liberate R.sub.2 H, and/or its mineral acid salt, and detecting the resulting R.sub.2 H in which R.sub.2 is as defined above; and the above reagent used therefor.