Facile structure determination of acylpolyamines, glutamatergic nerve blocker obtained from the venom of the Joro spider (Nephila clavata) was carried out with the use of micro-column LC/MS and high energy collision induced dissociation (CID) mass spectrometry. 6-hydroxyindole-3-acetyl was proposed previously as a putative partial structure, for the acyl moiety of hydroxyindole-type polyamines (NPTX-1 to -6). The NMR data obtained for NPTX-6, NPTX-687 and hydroxyindole-3-acetic acid which was released by acid hydrolysis of Nephila clavata crude venom extracts proved that the lipophilic head is the 4-hydroxyindole-3-acetic acid. Various hydroxyindole-3-acetyl polyamines were found in N. Clavata venom and characterized by mass spectrometry. As a result, type-E, a new class of generalized acylpolyamine structure was proposed in addition to the previously reported polyamine backbones type-A to -D.