This thesis consists of four Chapters unified by a singular theme – how do we develop disease models that faithfully reproduce the pathology seen in patients suffering from neurodegenerative disorders associated with the Tau protein, such as Alzheimer’s disease? To answer this question, we must first understand the underlying mechanisms leading to the formation and progression of Tau pathology in detail. Next, it is necessary to scrutinize the models that are being used for the research and development of therapies and drugs. And finally, it is important to discover novel approaches to study the pathological mechanisms that are occurring in the brain and develop models that are accurate and will have a high potential to lead to successful translation of the laboratory findings to the patients. In Chapter 1 “To target Tau pathologies, we must embrace and reconstruct their complexities” and Chapter 2 “Revisiting the grammar of Tau aggregation and pathology formation: How new insights from brain pathology are shaping how we study and target Tauopathies” we present the in-depth review of the Tau investigation field, and the in vitro and in cell models available. In Chapter 3 “ClearTau: a novel efficient method for production of pathology-resembling co-factor-free Tau fibrils” we present a novel, quick, cheap method for producing completely co-factor-free fibrils. We show that Tau fibrils generated using this method – ClearTau fibrils - exhibit amyloid-like features and morphological properties more reminiscent of the properties of the brain-derived Tau fibrils compared to the in vitro Tau fibrils produced by standard methods of preparation. The ClearTau fibrils retain the ability to bind RNA, share some structural/morphological features with brain-derived pathological fibrils and induce efficient seeding of Tau aggregation in vitro, in biosensor cells, and human neurons. As a proof of concept, we use the ClearTau assay as a platform for screening Tau aggregation modulator compounds. Finally, the generation of ClearTau fibrils also paves the way for a more systematic analysis of Tau post-fibrillization PTMs and fibril interactome without interference from heparin or heparin-induced polymorphisms. Our novel method for in vitro ClearTau fibril production represents a crucial milestone for developing accessible, cheap, and high-fidelity tools for studying the Tau fibrillization processes relevant to neurodegenerative diseases. Furthermore, these advances open new opportunities to investigate the pathophysiology of disease-relevant Tau aggregates and will facilitate the development of Tau pathology-targeting and modifying therapies and PET tracers that can distinguish between different Tauopathies. Finally, in Chapter 4 “What exactly are Tau biosensor assays sensing? In-depth characterization of the commonly used cellular model thought to report on the Tau seeding propensity” we are closely interrogating the widely-used cellular model.Collectively, this work presents a) the in-depth review of current knowledge and state of the art of Tau research; b) identifies shortcomings and gaps in research; c) presents a novel in vitro model to study Tau aggregation and d) scrutinizes the widely-used Tau cellular model and dives deep into the mechanisms of cell responses to Tau fibrillar seed.