We describe here a rapid method to determine the cyclic structure and disulfide linkages of highly stable cyclotides via a combination of flash partial acid hydrolysis, LC-MS/MS, and computational tools. Briefly, a mixture of closely related cyclotides, kalata B1 and varv A purified from Viola yedoensis was partially hydrolyzed in 2 M HCl for 5 min by microwave-assisted hydrolysis or for 30 min in an autoclave oven (121 degrees C and 15 psi). The partially hydrolyzed peptide mixture was then subjected to LC-MS/MS analysis, with the disulfide linked-peptides fragmented by collision activated dissociation (CAD). A computer program written in-house (available for download at http://proteomics.sbs.ntu.edu.sg/cyclotide_SS ) was used for interpreting LC-MS/MS spectra and assigning the disulfide bonds. Time-point analysis of single-disulfide fragments revealed that nonrandom acid catalyzed fragmentation mostly occurred at the turns which are solvent-exposed and often contain side chain functionalized amino acids such as Asx/Glx and Ser/Thr. In particular, the most susceptible bond for acid hydrolysis in kalata B1 and varv A was found to be the highly conserved N25-G26 which is also the head-to-tail ligation site of the linear precursor proteins, indicating that formation of the three disulfide bonds might precede cyclic structure closure by N25-G26 ligation. This observation is consistent with the recent report that the N25-G26 bond formation is the last step in the cyclotide biosynthetic pathway. The process demonstrated here can potentially be a high throughput method that is generally applicable to determine disulfide bonds of other relatively low-abundance cyclotides.