We report a state‐of‐the‐art spectroscopic study of an archetypical barbaralone, conclusively revealing the valence tautomerism phenomena for this bistable molecular system. The two distinct 1‐ and 5‐substituted valence tautomers have been isolated in a supersonic expansion for the first time and successfully characterized by high‐resolution rotational spectroscopy. This work provides irrefutable experimental evidence of the [3,3]‐rearrangement in barbaralones and highlights the use of rotational spectroscopy to analyze shape‐shifting mixtures. Moreover, this observation opens the window toward the characterization of new fluxional systems in the isolation conditions of the gas phase and should serve as a reference point in the general understanding of valence tautomerism.