Venom peptides are currently being developed as novel drugs and bioinsecticides. A large number of ant species have evolved a venom rich in peptides and proteins used for predation, defence and communication and therefore represent an untapped source of potential lead compounds, with only 72 currently described peptides. One aspect of ant venoms that has not been investigated is the difference in venom composition obtained using differing venom collection methods and the potential variations in venom composition between ant colonies of the same species. The present study therefore aimed to determine any differences in the peptide and protein components of the bullet ant (Paraponera clavata)collected by either manual venom gland dissection or electrical stimulation, as well as difference between ant venoms collected from different colonies. Venom proteins were separated by 2D-PAGE and analysed by nanoESI-MS. Peptidomic analyses were carried out by separating venoms on C18 RP-HPLC then analysing by MALDI-MS. The proteomic experiments revealed numerous proteins that were assigned a biological function (96 in total) of which 70% were common to both collection methods. The remaining 30% of proteins were either structural or metabolic proteins. The peptidomic analysis revealed a large number of peptides (309 in total) of which only 30% were common to both collection methods. This was far lower than what was observed from protein matches. Therefore, we have shown that each method reveals a unique set of peptides and proteins, however, extracting venom via electrical stimulation, which is not destructive to the ants, is the preferred method of collection as it contains all the major components. There were also notable differences in peptide and protein expression between the venoms obtained from the two different P. clavata colonies. These findings further demonstrate the richness and diversity of ant venoms as potential sources of bioactive compounds.