INTRODUCTION: Determining apolipoprotein E (APOE) ε4 allele status, a key genetic risk factor for Alzheimer's disease (AD), requires molecular genotyping infrastructure not widely accessible beyond specialized centers. METHODS: A fully automated high-throughput apoE E4 proteotyping immunoassay was evaluated for clinical performance (460 participants across three cohorts) and analytical validity. Concordance with polymerase chain reaction (PCR)-based genotyping and measures of analytical validity were reported. RESULTS: The apoE E4 immunoassay demonstrated 99.6% (95% confidence interval [CI]: 98.4% to 99.9%) concordance with PCR-based APOE ε4 genotype results across the pooled clinical cohort; 100.0% (95% CI: 97.1% to 100.0%) in those with AD (N = 127) and 99.4% (95% CI: 97.8% to 99.8%) in those without AD (333). The assay met analytical validity criteria for E4 isoform specificity, interference, precision, and stability. DISCUSSION: The apoE E4 immunoassay demonstrated high concordance with PCR-based genotyping and robust analytical validity, offering an accessible alternative for APOE ε4 zygosity assessment. HIGHLIGHTS: A novel high-throughput plasma-based proteotyping immunoassay for APOE ε4 zygosity classification was developed and evaluated for clinical performance and analytical validity. The apoE E4 immunoassay demonstrated high concordance (99.6%) with PCR-based APOE ε4 genotyping across a diverse international cohort, and a robust analytical profile. An apoE E4 immunoassay may offer a more cost-effective and accessible alternative to DNA genotyping approaches currently used for AD risk evaluation and anti-amyloid treatment decisions.