A multi-analytical investigation and ad hoc sampling method designed to support the conservation treatment of a 16th-century panel painting are described in this work.The painting, titled Madonna of the Tent and owned by the Musei Reali Torino (Turin, Italy), had been historically attributed to Raffaello Sanzio and subsequently identified as a copy after the Master – the original version of which is displayed at the Alte Pinakothek in Munich (Germany). The conservation treatment was recently carried out at Centro per la Conservazione ed il Restauro dei Beni Culturali “La Venaria Reale” (CCR), located in Venaria Reale (Turin, Italy). The work was particularly opaque and yellowed, suggesting the presence of different overlaying varnishes and, likely, overpainting.The scientific study of this work had two main aims: knowing the exact number and composition of the overlaying varnishes to select the best cleaning solution, and detecting the possible presence of overpainting, over the original stratigraphy.Coupling macro X-ray fluorescence scanning (MA-XRF), hypercolorimetric multispectral imaging (HMI), and UV–vis–NIR fiber optic reflectance spectroscopy (FORS) allowed for general mapping and characterization of color paints and pigments. Observing and analyzing some cross sections with an optical microscope under Vis and UV light and SEM/EDS was essential for understanding the stratigraphy. An ad hoc sampling method and further micro-invasive analyses were required for an accurate study of the chemical composition of the varnishes and their position in the stratigraphy. Especially the image of a cross section under UV light was a guide to selectively remove the scrapings for Fourier transform infrared spectroscopy (FTIR) and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) analyses; based on perceptible differences in UV-induced luminescence (UVL) of the layers, sampling of the varnishes was carried out, layer by layer, under a UV lamp.This work describes the complex process necessary to answer crucial questions in the conservation field. The adopted protocol exploited the full ability and complementarity of each imaging and spot technique used. The outcomes and methodological approach enabled us to trace the history of the past treatments and support the recent conservation treatment of the painting.