Establishing affordable, efficient, accessible, innovative, and multidisciplinary methodologies to the diagnosis of the conservation state of an artwork is key to carry out appropriate strategies of conservation and consequently to the creation of modern public policies on cultural heritage. Limited access to large-format paintings is a challenge to restoration scientists seeking to obtain information quickly, in a non-destructive and non-invasive manner, and identify regions of interest. Therefore, we put forward two unique software tools based on multispectral imaging techniques, with the long-term aim to assess the artist’s intentions, creative process, and colour palette. This development paves the way for a comprehensive and multidisciplinary understanding of the mysteries encompassed in each pictorial layer, through the study of their physical and chemical characteristics. We conducted the first ever study on Musas I and Musas II, two large-format paintings by Italian artist Carlo Ferrario, located in the National Theatre of Costa Rica. In this study, we used our novel imaging techniques to choose regions of interest in order to study sample layers; while also assessing the works’ state of conservation and possible biodeterioration. We explored the applications of our two versatile software tools, RegionOfInterest and CrystalDistribution, and confirmed paint stratigraphies by means of microscopy and spectroscopy analyses (OM, SEM-EDX, Fluorescent microscopy, FTIR-ATR and micro-Raman). In a pilot study, we identified the artist’s main colour palette: zinc white, lead white, chrome yellow, lead read, viridian, along with artificial vermilion and ultramarine pigments. We were able to identify artificial vermilion and ultramarine and distinguish them from the natural pigments using CrystalDistribution to map the average size and diameter of the pigment crystals within the paint layers. This study demonstrated that software-based multidisciplinary imaging techniques are novel in establishing preventive and non-invasive methods for historical painting conservation studies, in addition, this study provides tools with great potential to be used in the future in applications such as virtual restoration.