Directly observing individual protein molecules in action at high spatiotemporal resolution has long been a holy grail for biological science. This is because we long have had to infer how proteins function from the static snapshots of their structures and dynamic behavior of optical makers attached to the molecules. This limitation has recently been removed to a large extent by the materialization of high-speed atomic force microscopy (HS-AFM). HS-AFM allows us to directly visualize the structure dynamics and dynamic processes of biological molecules in physiological solutions, at subsecond to sub-100-ms temporal resolution, without disturbing their function. In fact, dynamically acting molecules such as myosin V walking on an actin filament and bacteriorhodopsin in response to light are successfully visualized. In this review, we first describe theoretical considerations for the highest possible imaging rate of this new microscope, and then highlight recent imaging studies. Finally, the current limitation and future challenges to explore are described.