Single-molecule methods have revolutionized scientific research by rendering the investigation of once-inaccessible biological processes amenable to scientific inquiry. Several of the more established techniques will be emphasized in this Review, including single-molecule fluorescence microscopy, optical tweezers, and atomic force microscopy, which have been applied to many diverse biological processes. Serving as a taste of all the exciting research currently underway, recent examples will be discussed of translocation of RNA polymerase, myosin VI walking, protein folding, and enzyme activity. We will end by providing an assessment of what the future holds, including techniques that are currently in development.