Abstract Background Antibody-based immunoassays have enabled quantification of very low concentrations of phosphorylated tau (p-tau) protein forms in cerebrospinal fluid (CSF), aiding in the diagnosis of AD. Mass spectrometry enables absolute quantification of multiple p-tau variants within a single run. The goal of this study was to compare the performance of mass spectrometry assessments of p-tau₁₈₁, p-tau₂₁₇ and p-tau₂₃₁ with established immunoassay techniques. Methods We measured p-tau₁₈₁, p-tau₂₁₇ and p-tau₂₃₁ concentrations in CSF from 173 participants from the TRIAD cohort and 394 participants from the BioFINDER-2 cohort using both mass spectrometry and immunoassay methods. All subjects were clinically evaluated by dementia specialists and had amyloid-PET and tau-PET assessments. Bland–Altman analyses evaluated the agreement between immunoassay and mass spectrometry p-tau₁₈₁, p-tau₂₁₇ and p-tau₂₃₁. P-tau associations with amyloid-PET and tau-PET uptake were also compared. Receiver Operating Characteristic (ROC) analyses compared the performance of mass spectrometry and immunoassays p-tau concentrations to identify amyloid-PET positivity. Results Mass spectrometry and immunoassays of p-tau₂₁₇ were highly comparable in terms of diagnostic performance, between-group effect sizes and associations with PET biomarkers. In contrast, p-tau₁₈₁ and p-tau₂₃₁ concentrations measured using antibody-free mass spectrometry had lower performance compared with immunoassays. Conclusions Our results suggest that while similar overall, immunoassay-based p-tau biomarkers are slightly superior to antibody-free mass spectrometry-based p-tau biomarkers. Future work is needed to determine whether the potential to evaluate multiple biomarkers within a single run offsets the slightly lower performance of antibody-free mass spectrometry-based p-tau quantification.