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• Non-invasive stratigraphy was successfully realized through SORS. • Layer composition in a fresco was determined. • Diagnosis and conservation tool was tested and validated for cultural heritage. We applied micro-scale Spatially Offset Raman Spectroscopy (micro-SORS) to perform the analysis of the layer composition in a fresco of the San Giuseppe church in Cagliari (XVIII century – under restoration). Standard Raman analysis of the surface evidenced the presence of expected pigments in the final plaster layer, such as hematite and calcium sulphate, beside the contribution of calcite. The second one is observed in its anhydrous form on the substrate layers (main Raman peak at 1024 cm−1) and in its hydrate form (1006 cm−1) from the surface. With micro-SORS analysis we evaluated the stratigraphy of the different layers of the plaster by monitoring the intensity ratio of the calcite and the anhydrous calcium sulphate. Four different layers with separation boundaries at about 80 μm, 600 μm and 2400 μm of defocusing distance were evidenced, corresponding to the painting layer "velo"(down to 80 μm), the plaster "rasatura" (down to 600 μm), the "arriccio" layer (down to 2400 μm) and the "rinzaffo" layer (below 2400 μm). The cross-sectional analysis by SEM imaging of a fragment from the fresco confirmed the micro-SORS findings, promoting the technique as a new non-invasive stratigraphy tool for cultural heritage. [ABSTRACT FROM AUTHOR]