Abstract INTRODUCTION Dementia is commonly caused by underlying pathologies driven by misfolded protein aggregates. Although dementia subtypes have distinct mechanisms, overlapping symptoms make diagnosis without biomarkers difficult. Misdiagnosis has previously hindered drug development by enrolling patients non‐specifically in trials. METHODS We developed a digital seed amplification assay (dSAA) that isolates individual aggregates in nanoliter compartments, enabling precise quantification of transactive response deoxyribonucleic acid binding protein 43 (TDP‐43) seeds in cerebrospinal fluid (CSF). RESULTS Testing 40 CSF samples from patients with genetic and sporadic frontotemporal lobar dementia with TDP (FTLD‐TDP), as well as healthy controls, we found elevated seed concentrations in FTLD‐TDP patients that correlated with disease severity, demonstrating the potential of dSAA as a sensitive diagnostic tool. DISCUSSION This study demonstrates a new quantitative, high‐sensitivity digital assay for TDP‐43 seeds in CSF. The platform's single‐aggregate resolution and low limits of detection and quantification establish a technical foundation for developing a diagnostic and monitoring tool for FTLD‐TDP and other TDP‐43–related diseases.