AbstractObjectivePlasma phosphorylated‐tau‐181 (p‐tau181) reliably detects clinical Alzheimer's disease (AD) as well as asymptomatic amyloid‐β (Aβ) pathology, but is consistently quantified with assays using antibody AT270, which cross‐reacts with p‐tau175. This study investigates two novel phospho‐specific assays for plasma p‐tau181 and p‐tau231 in clinical and asymptomatic AD.MethodsPlasma p‐tau species were quantified with Simoa in 44 AD patients, 40 spouse controls and an independent cohort of 151 cognitively unimpaired (CU) elderly who underwent Aβ‐PET. Simoa plasma Aβ42 measurements were available in a CU subset (N = 69). Receiver operating characteristics and Aβ‐PET associations were used to evaluate biomarker validity.ResultsThe novel plasma p‐tau181 and p‐tau231 assays did not show cross‐reactivity. Plasma p‐tau181 accurately detected clinical AD (area under the curve (AUC) = 0.98, 95% CI 0.95–1.00) as well as asymptomatic Aβ pathology (AUC = 0.84, 95% CI 0.76–0.92), while plasma p‐tau231 did not (AUC = 0.74, 95% CI 0.63–0.85 and 0.61, 95% CI 0.52–0.71, respectively). Plasma p‐tau181, but not p‐tau231, detected asymptomatic Aβ pathology more accurately than age, sex and APOE combined (AUC = 0.64). In asymptomatic elderly, correlations between plasma p‐tau181 and Aβ pathology were observed throughout the cerebral cortex (ρ = 0.40, p < 0.0001), with focal associations within AD‐vulnerable regions, particularly the precuneus. The plasma Aβ42/p‐tau181 ratio did not reflect asymptomatic Aβ pathology better than p‐tau181 alone.InterpretationThe novel plasma p‐tau181 assay is an accurate tool to detect clinical as well as asymptomatic AD and provides a phospho‐specific alternative to currently employed immunoassays.