Barrett’s esophagus (BE) is a known precursor to Esophageal Adenocarcinoma (EAC). Guidelines recommend BE screening in populations with multiple risk factors, for which non-endoscopic esophageal cell collection with biomarker testing is considered an acceptable alternative to esophagogastroduodenoscopy (EGD). This study presents analytical validation of EsoGuard® (EG), a two gene DNA methylation biomarker assay, in esophageal cell samples collected with the 510(k)-cleared, swallowable EsoCheck® (EC) device. EG is a Next Generation Sequencing (NGS) assay that evaluates methylated Vimentin (VIM) and Cyclin A1 (CCNA1) genes, clinically validated biomarkers for detection of BE and EAC. The studies were conducted according to standards of College of American Pathology (CAP), Clinical Laboratory Improvement Amendments (CLIA), and New York (NY) state requirements for analytical validation of molecular assays. Comparison to Sanger sequencing showed EG was 100% accurate at all 31 CpG sites evaluated by the assay. Analytical sensitivity, specificity and accuracy of the assay was 89%, 100% and 96% respectively. Intra- and inter-assay precision was 100%. Limit of detection (LOD) was 1 in 400 methylated cells and reference range was 84%. In summary, EsoGuard is a robust DNA methylation test to detect BE with high accuracy, repeatability, and reproducibility in samples collected using the EsoCheck device.