4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene derivatives (BODIPYs) are fluorescent organic dyes that are widely used as non-radioactive labels in biological analyses. The fragmentation behaviour of ten structurally related BODIPYs was studied using tandem mass spectrometry (MS/MS), to support the structural elucidation process during synthesis. The BODIPYs were investigated by electrospray ionization (ESI)-MS/MS, utilizing collision-induced dissociation (CID) data from triple quadrupole MS and high-resolution, accurate mass CID data from Fourier transform ion cyclotron resonance (FTICR) experiments. Unusual radical molecular cations ([M](+•)) were formed directly during the ESI process. These radical species dissociated into a large range of product ions during the subsequent CID experiments. Superimposed dissociations originating from parallel [M](+•) and [M+H](+) decompositions significantly complicated the interpretation of the MS/MS spectra. Detailed dissociation mechanisms were proposed in this study for BODIPY dyes. The elemental formulae of CID product ions were unambiguously assigned using FTICR-MS and unique fragment ions were discovered for the rapid identification of methyl, ethyl, butyl, tert-butyl, and phenyl substituents of individual dyes in BODIPY synthesis mixtures by low-resolution MS.