In the last decade, the hyperspectral imaging (HSI) method allowed performing non-invasive analysis in the field of cultural heritage. However, a considerable limitation was given by redundant and time-consuming features, with the necessary application of statistical algorithms and image-processing tools to extract relevant information. In this study, the Centro Nacional de Aceleradores (CNA) and the Group of Interdisciplinary Physics (GFI) of the School of Engineering (ETSI) of the University of Seville tested the application of three different hyperspectral cameras in the visible and near-infrared (VNIR) and short-wave infrared (SWIR) range for the investigation of an ancient painting. A reference-based procedure was realised to build a starting personal library and to evaluate the best working conditions for non-invasive and non-destructive characterisation with data treatment using the commercially available software Evince® and Specim IQ® to apply, respectively, the Principal Component Analysis (PCA) model functions and the classification method. The evaluation of the protocol was tested by acquiring complementary information by X-ray fluorescence (XRF), Ultraviolet Luminescence (UVL) imaging, and Infrared Reflectography (IRR). This exploration established a simplified protocol to analyse the large collection of paintings of the Archbishop’s Palace and the Cathedral of Seville.