Cilia and flagella are cell machines that power hydrodynamic forces by fast beating. They are composed of bundles of 9 + 2 microtubules that associate with several protein structures including axonemal dyneins and their regulators. Spermatozoa are single cells with in most cases a single flagellum and are good cell models to study how flagellar movements are regulated and linked to cell behavior. In addition, the assessment of sperm motility is an important diagnostic tool for evaluating male fertility in human reproductive medicine, and in livestock and fisheries sciences. Microscopic analyses of the movements of spermatozoa and their flagellar waveforms and propagation have been carried out using high-speed cameras and stroboscopic illumination. Computer-assisted sperm analysis (CASA) now comprises an automated set of methods to evaluate sperm quality for fertility. Here, we summarize the microscopy systems used for evaluating sperm motility, including CASA, and introduce updates on the molecular mechanism of flagellar movement and regulation that are linked to motility parameters. Furthermore, we introduce recent techniques employed to measure key factors controlling sperm motility.