This paper presents the correlative imaging of collagen denaturation by nonlinear optical microscopy (NLO) and nanoscale infrared (IR) spectroscopy to obtain morphological and chemical information at different length scales. Such multiscale correlated measurements are applied to the investigation of ancient parchments, which are mainly composed of dermal fibrillar collagen. The main issue is to characterize gelatinization, the ultimate and irreversible alteration corresponding to collagen denaturation to gelatin, which may also occur in biological tissues. Key information about collagen and gelatin signatures is obtained in parchments and assessed by characterizing the denaturation of pure collagen reference samples. A new absorbing band is observed near the amide I band in the IR spectra, correlated to the onset of fluorescence signals in NLO images. Meanwhile, a strong decrease is observed in Second Harmonic signals, which are a structural probe of the fibrillar organization of the collagen at the micrometer scale. NLO microscopy therefore appears as a powerful tool to reveal collagen degradation in a non-invasive way. It should provide a relevant method to assess or monitor the condition of collagen-based materials in museum and archival collections and opens avenues for a broad range of applications regarding this widespread biological material.In museums, libraries and archives, skin is largely present in collections as rawhide, parchment or leather since it has been used for utilitarian purposes, for example in clothes, vessels, writing supports and musical instruments or for decorative purposes. In Western Europe, parchment was the main writing support in the Middle Ages up to the growth of paper production in the 14-15 th centuries. Made from an untanned animal skin, it was preserved by liming, scraping and drying the skin under tension 1 . Compared to skin, parchment retains only the dermis layer, made mostly of fibrillar collagen (type I).Among conservators and scientists involved in the preservation of cultural heritage, gelatinization is a term commonly used for parchment alteration. It refers to the collagen denaturation to gelatin, where the triple helix is dissociated. Nevertheless, little is known about the actual transformation process in parchments and the influencing factors. UV radiation, heat or pH can cause perturbations in the bonding within and in-between collagen molecules but this might not be apparent unless water comes into contact with the material. In such a damaged structure, called a "pre-gelatinized" state, the access of water to the collagen molecules is considered to cause the loss of the hydrogen bonds holding the triple helix, resulting in the unfolding of the molecule. The gelatinization resulting from the action of water is accelerated by the additional energy provided by heating 2,3 . Given that gelatinization is irreversible, the major challenge for the conservation of parchment is to identify collagen in the "pre-gelatinized" state 2,3 so that suitable treatments or cons...