Valvasor's library is a unique example of a Seventeenth century personal library, including over 7300 prints and 1530 books. Today, it is kept in the Metropolitan Library of the Zagreb Archdiocese which is part of the Croatian State Archives. In this study, we analysed a selection of papers from Valvasor's collection (VC) of unknown origin and composition. We used a dual approach, combining the results of the analyses of two sets of samples to find the most useful tools for the characterization of historical handmade papers. 144 randomly selected papers were included in the study. As the VC is a cultural heritage, only non-destructive analyses were used, such as visual inspection, surface imaging, measurements of thickness and the determination of optical properties (brightness, yellowness, opacity and gloss). According to the optical properties, papers from the VC could be characterized as yellowish and opaque, without gloss. Due to the fact that the VC papers could not be destroyed, we took another set of samples consisting of 10 historical papers (HP) from archival materials, dating from the 16th to nineteenth century, and subjected them to non-destructive, as well as micro-destructive analyses. This was done to supplement the findings of non-destructive analyses of the original collection. Initially, spot tests were performed to determine water absorbency and to identify the presence of lignin and starch in the paper samples. SEM–EDS and XRF were applied for the identification of inorganic elements. FTIR analyses were used to identify the chemical structures of the paper components. Microscopic analyses were performed in two ways: the paper surface was imaged with a digital microscope, and the morphological characteristics of the fibres were studied using an optical microscope. The dominant fibres present were flax and hemp, with a smaller proportion of cotton. Additionally, thick and thin light brown fibres resembling straw were detected. The presence of calcium-containing components, gelatine and alum could be confirmed with SEM–EDS, XFR and FTIR analysis. Comparing the results of analyses performed on two sets of samples a connection was explored by which it was possible to define the characterization of VC papers. The main goal of the study was to document and characterize a specific collection for the future use of researchers of handmade paper or paper conservators. Furthermore, the study may serve as a model approach for other researchers who seek to document the characteristics of paper in their collections.