Striated muscle is an elegant system for study at many levels. Much has been learned about the mechanism of contraction from studying the mechanical properties of intact and permeabilized (or skinned) muscle fibers. Structural studies using electron microscopy, X-ray diffraction or spectroscopic probes attached to various contractile proteins were possible because of the highly ordered sarcomeric arrangement of actin and myosin. However, to understand the mechanism of force generation at a molecular level, it is necessary to take the system apart and study the interaction of myosin with actin using in vitro assays. This reductionist approach has lead to many fundamental insights into how myosin powers muscle contraction. In addition, nature has provided scientists with an array of muscles with different mechanical properties and with a superfamily of myosin molecules. Taking advantage of this diversity in myosin structure and function has lead to additional insights into common properties of force generation. This review will highlight the development of the major assays and methods that have allowed this combined reductionist and comparative approach to be so fruitful. This review highlights the history of biochemical and biophysical studies of myosin and demonstrates how a broad comparative approach combined with reductionist studies have led to a detailed understanding of how myosin interacts with actin and uses chemical energy to generate force and movement in muscle contraction and motility in general.