The variations of the fluorescence emission of carmine lake travelling through an absorbing and scattering medium, such as a paint layer, were investigated by ultraviolet (UV)-visible absorption, fluorescence spectroscopy, and imaging techniques. Samples of the lake were studied in dilute and saturated solutions, on a reference test panel and a real case study. Relevant spectral modifications have been observed as a function of the lake concentration mainly consisting of a fluorescence quenching, red shift of emission maxima, and deformation of emission band. The application of a correction factor based on the Kubelka-Munk model allowed fluorescence spectra obtained in solution and on painted samples of known composition to be compared and correlated, highlighting that the fluorescence of the lake within paint layers is affected by both self-absorption and aggregation phenomena. This approach has been successfully applied on a painting by G. Vasari for the noninvasive identification of carmine lake. The results reported here emphasize the necessity of taking physical phenomena into account in the interpretation of the fluorescence spectra for a proper and reliable characterization and identification of painting materials in works of art.