A painting from the Botticelli workshop has been studied after its recent discovery, with an innovative and noninvasive approach combining two‐ dimensional scanning macro‐x‐ray fluorescence imaging (MA‐XRF) and a laboratory‐based depth‐resolved site‐selective confocal micro‐x‐ray fluorescence (CXRF) device. These analyses were supported by measurements on cross‐sections taken from the artwork using scanning electron microscopy coupled with an energy‐dispersive x‐ray system. The aims of this study are to confirm the painting's attribution and authentication, find characteristic markers and features, understand the artist's technique, materials used, and palette, and all of it while reducing sampling. The analyzing approach used combines imaging and site‐selective techniques while avoiding, reducing and replacing sampling without compromising the results. Chemical maps of the painting were obtained by MA‐XRF and enabled the identification of zones, colors, and chemical elements of interest alongside with a first assumption on the pigments used. Depth profiles were then performed in precise areas and colors using CXRF, allowing to evidence overlaying paint layers and obtain a more complete 3D vision of the painting. Contrasting the findings using this new methodology with the traditionally employed analysis process involving microsampling allowed us to determine the accuracy and veracity of our conclusions.