Structural biology is currently undergoing a transformation into dynamic structural biology, which reveals the dynamic structure of proteins during their functional activity to better elucidate how they function. Among the various approaches in dynamic structural biology, high-speed atomic force microscopy (HS-AFM) is unique in the ability to film individual molecules in dynamic action, although only topographical information is acquirable. This review provides a guide to the use of HS-AFM for biomolecular imaging and showcases several examples, as well as providing information on up-to-date progress in HS-AFM technology. Finally, we discuss the future prospects of HS-AFM in the context of dynamic structural biology in the upcoming era. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.