Mechanisms of collision induced dissociation (CID) under the conditions of electrospray ionization mass spectrometry (ESI-MS) of cyclic-di-adenosine mono phosphate (c-di-AMP) and cyclic-di-guanosine mono phosphate (c-di-GMP) are described. The CID mass spectrum of doubly protonated ([M+2H]2+) c-di-AMP differs from that of singly protonated ([M+H]+) and the mechanisms of dissociation of these two ionic forms are proposed. CID of singly deprotonated form ([M-H]-) of c-di-AMP was also carried out and its fragmentation mechanism is delineated. Ring-opening step seems to be imperative and foremost prior to subsequent dissociation events, during the fragmentation of both cationic and anionic forms of c-di-AMP. In the case of CID of [M+H]+ of c-di-GMP, the abundance of fragment ions indicate that ring-opening mediated fragmentation may not be a favored pathway, while loss of guanine may be relatively more preferred. Interestingly, fragmentation of [M-H]- of c-di-GMP appears to be very similar to that of c-di-AMP, which involves ring-opening step, suggesting that nitrogen base does not influence CID pathways of [M-H]- species. Understanding such dissociation mechanisms will prove useful for discovery and validation of novel hybrid cyclic dinucleotides and their analogues. Also, such comprehensions will be helpful in distinguishing different isobaric molecules possessing varied molecular structures. © 2014 Elsevier B.V.