This study compares the performance of mass spectrometry and immunoassays in quantifying phosphorylated tau (p-tau) proteins (p-tau181, p-tau217, and p-tau231) in cerebrospinal fluid (CSF) for the diagnosis of Alzheimer's disease (AD). The research involved 173 participants from the TRIAD cohort and 394 participants from the BioFINDER-2 cohort. Both methods were used to measure p-tau concentrations, and the results were compared using Bland–Altman analyses and ROC curve analyses. The study found that while mass spectrometry and immunoassays showed high agreement for p-tau217, p-tau181 and p-tau231 measured using antibody-free mass spectrometry had lower performance compared to immunoassays. Specifically, p-tau181 quantified using mass spectrometry had significantly lower diagnostic accuracy for amyloid-PET positivity than when measured using immunoassays. The study suggests that while both methods are effective, immunoassays may be slightly superior for p-tau181 quantification. However, mass spectrometry's ability to quantify multiple analytes in a single run could be advantageous for comprehensive AD biomarker assessment.This study compares the performance of mass spectrometry and immunoassays in quantifying phosphorylated tau (p-tau) proteins (p-tau181, p-tau217, and p-tau231) in cerebrospinal fluid (CSF) for the diagnosis of Alzheimer's disease (AD). The research involved 173 participants from the TRIAD cohort and 394 participants from the BioFINDER-2 cohort. Both methods were used to measure p-tau concentrations, and the results were compared using Bland–Altman analyses and ROC curve analyses. The study found that while mass spectrometry and immunoassays showed high agreement for p-tau217, p-tau181 and p-tau231 measured using antibody-free mass spectrometry had lower performance compared to immunoassays. Specifically, p-tau181 quantified using mass spectrometry had significantly lower diagnostic accuracy for amyloid-PET positivity than when measured using immunoassays. The study suggests that while both methods are effective, immunoassays may be slightly superior for p-tau181 quantification. However, mass spectrometry's ability to quantify multiple analytes in a single run could be advantageous for comprehensive AD biomarker assessment.