Intracellular patch-clamp electrophysiology, one of the most ubiquitous, high-fidelity techniques in biophysics, remains laborious and low-throughput. While previous efforts have succeeded at automating some steps of the technique, we have created a robotic ‘PatcherBot’ system that can perform many patch-clamp recordings sequentially, fully unattended. Comprehensive automation is accomplished by outfitting the robot with machine vision, and cleaning pipettes instead of manually exchanging them. The PatcherBot can obtain data at a rate of 16 cells per hour and work with no human intervention for up to 3 h. We have demonstrated the broad applicability and scalability of this system by performing hundreds of recordings in tissue culture cells and mouse brain slices with no human supervision. The system is potentially transformative for applications that depend on many high-quality measurements of single cells, such as drug screening, protein functional characterization, and multimodal cell type investigations.