Correct cell/cell interactions and motion dynamics are fundamental in tissue homeostasis, and defects in these cellular processes cause diseases. Therefore, there is strong interest in identifying factors, including drug candidates that affect cell/cell interactions and motion dynamics. However, existing quantitative tools for systematically interrogating complex motion phenotypes in timelapse datasets are limited. We present Motion Sensing Superpixels (MOSES), a computational framework that measures and characterises biological motion with a unique superpixel ‘mesh’ formulation. Using published datasets, MOSES demonstrates single-cell tracking capability and more advanced population quantification than Particle Image Velocimetry approaches. From > 190 co-culture videos, MOSES motion-mapped the interactions between human esophageal squamous epithelial and columnar cells mimicking the esophageal squamous-columnar junction, a site where Barrett’s esophagus and esophageal adenocarcinoma often arise clinically. MOSES is a powerful tool that will facilitate unbiased, systematic analysis of cellular dynamics from high-content time-lapse imaging screens with little prior knowledge and few assumptions.