An integrated method of ion mobility mass spectrometry and high-performance liquid chromatography (HPLC) has been used to investigate the isomeric distribution of a popular fluorescent dye DCM (4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran) in methanol solution. Chromatographic separation of DCM isomers in methanol has been performed by probing the molecular mass (DCMH+), and two distinctly separated peaks are observed at retention times 3.73 (peak-I) and 3.87 (peak-II) min, where the latter one appears nearly twice as intense as the former. However, peak-I appears much weaker compared to peak-II if the chromatogram is recorded by optical probing at the absorption maximum of this dye (467 nm). The ion mobility (IM) spectra of DCMH+ ions corresponding to each of the LC-separated factions show three common peaks A, B, and C, with collision cross-section (CCS) values of 174, 185, and 197 angstrom(2), respectively, but their relative intensities in the two IM spectra appear in opposite sequences. The three IM peaks have been assigned by considering the theoretically calculated CCS values of 13 possible isomers of DCMH+ ions. The IM spectral features also reveal that isomeric interconversions occur during the ESI process. Electronic structure calculations have been used to optimize the geometries of the four isomers of solvated DCM and the corresponding protomeric structures of DCMH+. The isomerization pathways and associated energy barriers have also been calculated. The gas-phase protomers are found to follow a completely different sequence of stability as compared to the neutral isomers. The analysis reveals that peak-I corresponds to one of the cis isomers, whereas peak-II arises due to cumulative contributions of the other three isomers. The absorption spectrum of DCM in methanol is simulated from the computed spectral profiles of the isomers which indicates a distribution of trans1, trans2, cis1, and cis2 isomers as 33.5, 61.5, 2.0, and 3.0%, respectively. The fragmentation behavior of DCMH+ ions in a collision-induced dissociation experiment has been found to be isomer dependent.