The research results presented in this paper are part of a larger study on the materials and techniques used in polychrome altarpieces of gilded woodcarving decoration (talha dourada) in Portugal. The paper focuses on a narrative Portuguese Altarpiece from Miranda do Douro, considered one of the masterpieces of talha dourada among all the retables of the Iberian Peninsula in XVIIth and XVIIIth centuries. Although on the Portuguese territory, the altarpiece was made by artists from the Royal Spanish school of Valladolid, under a mannerist style. Thus the study opens a window on the artists' circulation between Spain and Portugal and influences of the Spanish schools in Baroque epoch on the Portuguese talha. During its history this altarpiece underwent several transformations and extensive conservation treatments in 1989. On this occasion more than 50 samples were collected and analyzed using an interdisciplinary multi-technique methodology. 27 of these samples are chosen for this study in order to investigate the chromatic palette, the materials and techniques used in the polychromy of the retable. A novel protocol of investigation using different conventional and unconventional analytical techniques (OM + fluorescent staining tests on cross-sections, Raman microscopy, XRD, XRF, X-ray micro-CT, SEM-EDX, MALDI-TOF-MS and LC-MS/MS) was established within an innovative research project (http://sites.fct.unl.pt/gilt-teller/) and applied on these samples. This protocol is necessary to confirm the results obtained in the 1989 campaign and to have further insight into the gilding and polychrome decoration materials and techniques and the additional information reported in the historical documents. The material and technical history of this important altarpiece will be thus re-documented from a scientific perspective, meant to confirm and bring new information on the decorative technique used in the creation of this complex Portuguese monument. Microsc. Res. Tech. 76:733-743, 2013.