This study presents the development of two novel Simoa immunoassays for measuring CSF p-tau205 and p-tau202, key markers of tau pathology in Alzheimer's disease (AD). The assays were validated across three independent cohorts: a discovery cohort (n=47), an unselected clinical cohort (n=212), and a research cohort (n=262) well-characterized by fluid and imaging biomarkers. CSF p-tau205 levels increased progressively across the AD continuum, while CSF p-tau202 was increased only in AD and amyloid (Aβ) and tau pathology positive (A+T+) cases. Both biomarkers showed stronger associations with tau-PET than Aβ-PET, and CSF p-tau205 was significantly associated with Braak V–VI tau-PET regions. CSF p-tau205 and p-tau202 were also associated with brain atrophy and cognition. In terms of diagnostic performance, CSF p-tau205 showed higher accuracy than p-tau202 in distinguishing AD cases. The study concludes that CSF p-tau205 is a promising biomarker for in vivo quantification of tau pathology in AD, and a potentially cost-effective alternative to tau-PET in clinical settings and clinical trials. The results suggest that CSF p-tau205 is more closely associated with tau pathology than Aβ pathology, and that it has a stronger association with tau-PET than Aβ-PET. The study also highlights the importance of tau pathology in the progression of AD and the need for fluid biomarkers that can specifically reflect and track aggregated tau pathology in the brain.This study presents the development of two novel Simoa immunoassays for measuring CSF p-tau205 and p-tau202, key markers of tau pathology in Alzheimer's disease (AD). The assays were validated across three independent cohorts: a discovery cohort (n=47), an unselected clinical cohort (n=212), and a research cohort (n=262) well-characterized by fluid and imaging biomarkers. CSF p-tau205 levels increased progressively across the AD continuum, while CSF p-tau202 was increased only in AD and amyloid (Aβ) and tau pathology positive (A+T+) cases. Both biomarkers showed stronger associations with tau-PET than Aβ-PET, and CSF p-tau205 was significantly associated with Braak V–VI tau-PET regions. CSF p-tau205 and p-tau202 were also associated with brain atrophy and cognition. In terms of diagnostic performance, CSF p-tau205 showed higher accuracy than p-tau202 in distinguishing AD cases. The study concludes that CSF p-tau205 is a promising biomarker for in vivo quantification of tau pathology in AD, and a potentially cost-effective alternative to tau-PET in clinical settings and clinical trials. The results suggest that CSF p-tau205 is more closely associated with tau pathology than Aβ pathology, and that it has a stronger association with tau-PET than Aβ-PET. The study also highlights the importance of tau pathology in the progression of AD and the need for fluid biomarkers that can specifically reflect and track aggregated tau pathology in the brain.