A combination of high-performance liquid chromatography (HPLC) and atmospheric-pressure chemical ionization mass spectrometry (APCI-MS and APCI-MS/MS) was used to detect and characterize linear polyamine derivatives in the venom of the spiders Agelenopsis aperta, Hololena curta and Paracoelotes birulai. The compounds were identified with a template approach, by which the collision-induced dissociation (CID) spectra of known compounds are directly compared and correlated with those of the analytes. To facilitate the perception of the spectra and the recognition of the structural features of the analytes, an ion nomenclature closely leaned on the accepted nomenclature for fragment ions of peptides or nucleic acids is introduced. The structure identification of polyamine derivatives by direct correlation of MS spectra is possible because such compounds show very distinctive fragmentation behavior. Of particular relevance is the fact that the signal patterns that are observed with analytes possessing different polyamine backbones are not only distinct with regard to mass distributions but also with regard to relative signal intensities, resulting in fingerprint-like signal patterns. The direct correlation of these patterns—more than the correlation of the ion distributions—was found to be of key significance. With this, the new approach is fundamentally different from the sequencing of peptides or nucleic acids, which are largely based on mass distributions. The method is more efficient and more reliable than the de novo interpretation of the MS data and it even allows the identification of polyamine portions in compounds that are analyzed within mixtures.