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Protein motion is defined by a network of interactions within the cellular environment, serving as a direct report of activity and function. The key challenges in single particle tracking (SPT) studies in live cells remain the assignment of biological meaning from a set of molecule trajectories, as well as the technical challenges in generating the volume of data necessary to characterize interaction pathways. Through advancements in optical engineering, automation, and computation, we have developed an industrial-scale system to track fast-moving proteins across thousands of conditions in mammalian cell lines. We applied this system to study the estrogen receptor (ER), a protein well known for its role in normal human development as well as its prominent role in many breast cancers. We present a chemical genetics screen of thousands of known bioactive compounds to identify novel ER modulators. Intriguingly …