The structure and energetic properties of four common oxazine dyes, Nile red, Nile blue A, Cresyl violet, and Brilliant cresyl blue, have been probed using a combination of infrared multiple-photon dissociation (IRMPD) spectroscopy and quantum chemical calculations. IRMPD spectra of the protonated dyes, as generated from an electrospray ionization (ESI) source, were collected in the range of 900-1800 cm(-1). Vibrational band assignments related to carbonyl and substituted-amine stretches were established from a comparison of the experimental spectra of these related systems as well as from a comparison with spectra generated by density functional theory (DFT) calculations. For Nile red, the thermochemical landscape for protonation at different basic sites was probed using DFT; comparison of IRMPD and calculated IR spectra reveals the site of protonation to be at the carbonyl oxygen. The structural information obtained here in the gas phase pertaining to these important fluorophores is anticipated to provide further insight into their associated intrinsic fluorescent properties in solution.