Investigations of wall paintings by Masolino da Panicale in the Baptistery of Castiglione Olona were conducted using a multi-analytical approach, based on in situ fluorescence imaging, multispectral reflectance imaging and non-destructive analysis of a historical cross-section. Fluorescence imaging of wall paintings, mainly focused on the discrimination and identification of organic compounds, has been conducted with a novel approach, which incorporates the use of spectrally-resolved and lifetime-resolved imaging devices, applied together for the first time for the analysis of paintings. Analysis of the UV-induced emissions from the surface of the paintings allowed the documentation of the selective use of a carmine-based red lake, identified using micro-Raman spectroscopy and Scanning Electron Microscopy. Visible reflectance measurements highlighted the distribution and use of different inorganic pigments. Finally, the in situ study, with the support of early 20th century photographs, provided additional information on Masolino’s style and revealed details and decorative elements of the painting, which are indiscernible under standard illumination.