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Fibrosis plays an important role in many different pathologies. It results from tissue injury, chronic inflammation, autoimmune reactions and genetic alterations, and it is characterized by the excessive deposition of extracellular matrix components. Biopsies are routinely employed for fibrosis diagnosis, but they suffer from several drawbacks, including their invasive nature, sampling variability and limited spatial information. To overcome these limitations, multiple different imaging tools and technologies have been evaluated over the years, including X-ray imaging, computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET) and single-photon emission computed tomography (SPECT). These modalities can provide anatomical, functional and molecular imaging information which is useful for fibrosis diagnosis and staging, and they may also hold potential for the longitudinal assessment of therapy responses. Here, we summarize the use of non-invasive imaging techniques for monitoring fibrosis in systemic autoimmune diseases, in parenchymal organs (such as liver, kidney, lung and heart), and in desmoplastic cancers. We also discuss how imaging biomarkers can be integrated in (pre-) clinical research to individualize and improve anti-fibrotic therapies.