<p>Transthyretin (TTR) is a circulating protein biomarker central to the evaluation of therapies for transthyretin amyloidosis. Clinical trials with Eplontersen, a TTR-targeting antisense oligonucleotide, required an assay with a context of use differing from existing assay, requiring a more sensitive method. This study describes the development, optimization, and validation of a quantitative immunoassay for TTR, guided by a context-of-use framework. Method development focused on calibration curve modeling, weighting, parallelism, and determination of the minimal required dilution. Antibody selection and calibrator characterization followed established best practices. Pre-validation experiments optimized antibody concentrations and curve-fitting (five-parameter logistic with 1/y² weighting). Assay performance was then evaluated through validation, including assessments of accuracy, precision, parallelism, specificity, stability, interference of endogenous binding partners, and comparison against other commercially available assays. The assay demonstrated a functional lower limit of quantitation of approximately 0.07&#xa0;mg/dL, which would enable measurement of &gt; 95% reductions in TTR after treatment. Parallelism improved at higher dilutions. The assay showed robust performance across endogenous samples, recombinant TTR variants and in the presence of potential interferents. Total analytical variability was shown to be 29.4%. Comparisons with commercial assays showed strong correlation, though absolute values varied by platform. This work establishes a robust, context-driven assay for quantifying TTR in clinical studies. The development process highlights generalizable principles in biomarker assay design, including matrix considerations, variability analysis, and curve optimization. These insights provide a transferable framework for biomarker method development in regulated environments.</p>