Contryphans, peptides containing a single disulfide bond, are found abundantly in cone snail venom. The analysis of a large dataset of available contryphan sequences permits a classification based on the occurrence of proline residues at positions 2 and 5 within the macrocyclic 23-membered disulfide loop. Further sequence diversity is generated by variable proteolytic processing of the contryphan precursor proteins. In the majority of contryphans, presence of Pro at position 2 and a D-residue at position 3 leads to a slow conformational dynamics, manifesting as anomalous chromatographic profiles during LC analysis. LC-MS analysis of diverse contryphans suggests that elution profiles may be used as a rapid diagnostic for the presence of the Pro2-Xxx3 motif. Natural sequences from C.inscriptus and C.frigidus together with synthetic analogs permit the delineation of the features necessary for abnormal chromatographic behaviour. A diagnostic for the presence of Pro at position 5 is obtained by the observation of non-canonical fragment ions, generated by N-C bond cleavage at the dehydroalanine residue formed by disulfide cleavage. Anomalous LC profiles supports Pro at position 2, while non-canonical mass spectral fragments established Pro at position 5, providing a rapid method for contryphan analysis from LC-ESI-MS/MS profiles of crude Conus venom. SIGNIFICANCE: Contryphans are peptides, widely distributed in cone snail venom, which display extensive sequence diversity. Heterogeneity of proteolytic processing of contryphan precursor proteins, together with post-translational modifications contributes to contryphan diversity. Contryphans, identified by a combination of mass spectrometry and transcriptomic analysis, are classified on the basis of sequence features, primarily the number of proline residues within the disulfide loop. Conformational diversity arises in contryphans by cis-trans isomerization of Cys-Pro bonds, resulting in characteristic chromatographic profiles, permitting identification even in crude venom mixtures. Rapid identification of contryphans in cone snail peptide libraries is also facilitated by diagnostic mass spectral fragments arising by non-canonical cleavage of the N-C bond at Cys(6).