The abnormal aggregation of the microtubule-associated protein Tau into neurofibrillary tangles (NFTs) is a hallmark of tauopathies including Alzheimer’s disease (AD). Tau pathology spreads along connected neural networks through the intercellular transfer of pathogenic Tau species, termed seeds. In a prion-like manner, seeds are secreted into the extracellular space and subsequently enter neighboring cells. Upon internalization, seeds nucleate the fibrillization of endogenous Tau via direct protein-protein contact. The highly conserved serine protease HTRA1 was shown to degrade Tau fibrils by disintegrating their core structure and proteolyzing extracted polypeptides in vitro. Further, HTRA1 inversely correlates with the occurrence of NFTs in AD brains. Based on these findings, the role of HTRA1 in the progression of tauopathies was addressed in this work with particular regard to Tau spreading. In order to elucidate the HTRA1 functionality in Tau pathology on a cellular level, a novel assay was applied. It relies on HEK293 cells overexpressing Tau tagged to CFP or YFP, respectively. Intracellular aggregation in these Biosensor cells was induced by the addition of Tau seeds. Aggregation of Tau results in close proximity of fluorescent tags and thus a FRET signal quantifiable by flow cytometry. HTRA1 is secreted, but its internalization was demonstrated in a plethora of cell lines, allowing cellular treatment by medium supplementation. Increasing HTRA1 amounts resulted in a FRET signal reduction in Biosensor cells and thus dissociation of intracellular Tau aggregates in a concentration-dependent manner. Treatment with HTRA1 prior to seed addition caused an even more significant decline of the FRET signal, suggesting inhibition of seeding. The effect of HTRA1 on Tau spreading was examined in a newly established in cellulo approach. HEK293 cells harboring Tau aggregates were indirectly cocultured with untreated Biosensor cells. Joint cultivation resulted in the emergence of a FRET signal indicating spreading of Tau seeds between the two cell populations. Overexpression of HTRA1 in donor cells or its addition to the medium reduced spreading. These data point to an antagonizing role of HTRA1 in the propagation of Tau pathology. In AD patients and tauopathy mouse models HTRA1 protein levels are up-regulated. Presumably, intracellular Tau aggregation enhances the secretion of HTRA1 that subsequently enters adjacent cells to impede seeding. Spreading of the protease was explored by the indirect co-culture of HTRA1 overexpressing cells and SH-SY5Y neuroblastoma or HEK293T cells. Both recipient cell lines were shown to internalize secreted HTRA1. Concomitant addition of Tau seeds and HTRA1 to cells revealed colocalization indicating a mutual uptake mechanism. Indeed, endocytosis was demonstrated for HTRA1, which is also a major internalization pathway of spreading Tau seeds. Further, co-cultivation of HTRA1 overexpressing cells with Biosensor cells harboring insoluble Tau resulted in disassembly of aggregates. In a similar assay, dissociation of Tau seeds mediated by HTRA1 spreading was shown in SH-SY5Y cells. In the process of Tau degradation, HTRA1 might be allosterically activated by the cysteine protease Calpain 2. Co-localization assays conducted herein suggest the interaction of both proteases and thus allow first insights into the regulation of HTRA1 in tauopathies. Cell viability assays demonstrated the HTRA1-mediated rescue of SH-SY5Y cells affected by toxic Tau seeds. Interaction of HTRA1 and pathogenic Tau species observed in patient-derived neurons further promotes the physiological relevance of HTRA1 in tauopathies. In the course of this work, HTRA1 was shown to hamper Tau spreading by dissociating Tau aggregates in donor cells, reducing Tau seeds and seeding in recipient cells. Moreover, Tau pathology potentially triggers spreading of HTRA1 itself to protect neighboring cells. These findings indicate a yet unknown defense mechanism that provides new opportunities for the treatment of tauopathies.