A new strategy to characterize glutaminergic blocker acylpolymines stored in a spider venom with mass spectrometry is described. The crude spider venom extracts are amenable to direct MALDI mass spectrometry analysis which provides a rapid and accurate means of measuring the molecular weights of acylpolyamines without the isolation of individual samples. Compared with the previously developed mu-column HPLC/MS method, this procedure provides more efficient detection and identification of complex venom constituents. Twenty-five acylpolyamines were detected from Brazilian garden spider Nephilengys cruentata crude venom extracts by both HPLC/MS and MALDI-mass spectrometry. These acylpolyamine structures were determined by high-energy collision induced dissociation MS/MS method. Most of the compounds were classified into the previously reported generalized structures types A to D, which were found from the venom of Nephilengys borbonica. The structures of four acylpolyamines (M + H)+, m/z 623, 646, 688, and 745, which were not contained in the venom of Nephilengys borbonicare were determined to have arginine at the polyamine chain terminal and were named NPTX-622, -645, -687, and -744, respectively.