Self-propagating, amyloidogenic mutant huntingtin (HTT) aggregates may drive progression of Huntington’s disease (HD). Here, we report the development of a FRET-based HTT aggregate seeding (FRASE) biosensor assay that enables the quantification of mutant HTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knock-in mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that HSA is high in fractions that contain small HTT fibrils but is low in fractions with large, insoluble HTT aggregates, indicating that small rather than large mutant HTT structures possess HSA. Finally, we assessed the neurotoxicity of mutant HTT seeds in an inducible Drosophila model transgenic for HTT. We found a strong correlation between HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mutant HTT structures with severe phenotypic consequences in vivo.