The disulfide-intact and disulfide-reduced β-subunit of Shiga toxin 2 (β-Stx2) from Escherichia coli O157:H7 (strain EDL933) has been identified by matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomic analysis using software developed in-house. E. coli O157:H7 was induced to express Stx2 by culturing on solid agar media supplemented with 10-50 ng mL(-1) of ciprofloxacin (CP). Bacterial cell lysates at each CP concentration were analyzed by MALDI-TOF-MS. A prominent ion at mass-to-charge (m/z) ~7820 was observed for the CP concentration range: 10-50 ng mL(-1), reaching a maximum signal intensity at 20 ng mL(-1). Complex MS/MS data were obtained of the ion at m/z ~7820 by post-source decay resulting in top-down proteomic identification as the mature, signal peptide-removed, disulfide-intact β-Stx2. Eight fragment ion triplets (each spaced Δm/z ~33 apart) were also observed resulting from backbone cleavage between the two cysteine residues (that form the intra-molecular disulfide bond) and symmetric and asymmetric cleavage of the disulfide bond. The middle fragment ion of each triplet, from symmetric disulfide bond cleavage, was matched to an in silico fragment ion formed from cleavage of the backbone at a site adjacent to an aspartic acid or glutamic acid residue. The flanking fragment ions of each triplet, from asymmetric disulfide bond cleavage, were not matched because their corresponding in silico fragment ions are not represented in the database. Easier to interpret MS/MS data were obtained for the disulfide-reduced β-Stx2 which resulted in an improved top-down identification.