Chronic, low-dose UV irradiation (UVR) leads to premature ageing of the skin characterised by wrinkles and loss of elasticity. Although the aged appearance of photoexposed skin may take years to manifest, the biochemical and molecular damage that underpins photoageing can occur following a single physiological dose of UVR. Understanding the damage mechanisms that are elicited in skin following acute UVR could help to inform strategies to delay photoageing. Targeted approaches show that UVR alters the abundance and structure of selected ECM proteins and protein assemblies, and these processes are driven by UV-induced reactive oxygen species (ROS). However, due to limitations in proteomic analysis, it has been difficult to evaluate the effects of acute UVR on an entire ECM-enriched proteome, containing hundreds of proteins. Peptide location fingerprinting (PLF) is a proteomic analysis tool that can identify structural changes in hundreds of proteins across different treatment groups using a non-targeted approach. The aim of this study was to use PLF to identify proteins within an ECM-enriched proteome that is structurally altered in response to UV. To confirm the utility of PLF, structural changes in native type-I collagen, known to be UV-resistant, and purified human fibronectin, known to be UV susceptible, were evaluated following UVR at 50 mJ/cm2 and 500 mJ/cm2. Results show that native type-I collagen displays no significant structural changes in response to UVR, whereas UV irradiated human tissue fibronectin shows significant structural changes within the functional domains binding fibrin/heparin and collagen. Using a ECM-enriched proteome in vitro, PLF identifies 25 proteins from amongst a proteome of 977 proteins that show structural changes in response to acute UVR (100 mJ/cm2). The highly variable UV chromophore content among this group of 25 proteins suggests that abundance of UV chromophores alone cannot account for the structural changes observed in response to UVR. Protein interaction analysis (STRING) shows that UV targeted proteins are involved in collagen fibril formation, glycosaminoglycan metabolism and protein translation, and assessment of protein domain organization (Uniprot) shows that acute UV alters domains involved in ECM binding (COL5A1, versican) and myosin motility. In conclusion, this study identifies a select group of extracellular and intracellular dermal proteins that are susceptible to acute UVR and identifies the specific protein domains in which acute UV-mediated damage occurs.