Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common techniques include optical tweezers, magnetic tweezers, and atomic force microscopy (AFM). These techniques are described and illustrated with examples highlighting current capabilities and limitations. Optical tweezers are versatile and can exert forces up to 100 pN while measuring displacement with sub-nanometer accuracy and sub-millisecond time resolution. They are used to study a wide range of biological processes, from cellular motility to DNA replication and segregation. Key components include a trapping laser and a high numerical aperture (NA) microscope objective. Position detection methods, such as back focal plane (BFP) interferometry, are crucial for accurate measurements. Magnetic tweezers are simpler to implement and can exert forces up to 1 nN. They are particularly useful for studying nucleic acid enzymes, such as DNA topoisomerases, due to their ability to apply infinite bandwidth, passive force clamping over large displacements. Magnetic tweezers are selective and insensitive to sample preparation, making them suitable for complex, heterogeneous environments. AFM is another technique used in single-molecule force spectroscopy, offering high-resolution imaging and force measurements. It is particularly useful for studying the mechanical properties of biological macromolecules and the interactions between them. The choice of technique depends on the specific application, with each technique having its strengths and limitations. Optical tweezers are versatile but can be affected by sample heating and photodamage, while magnetic tweezers are selective and insensitive to sample preparation but have limited force and displacement ranges. AFM is highly resolving but more complex and expensive to set up. Overall, single-molecule force spectroscopy techniques have revolutionized the study of biological processes by providing precise and detailed measurements of forces and motions at the molecular level.Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common techniques include optical tweezers, magnetic tweezers, and atomic force microscopy (AFM). These techniques are described and illustrated with examples highlighting current capabilities and limitations. Optical tweezers are versatile and can exert forces up to 100 pN while measuring displacement with sub-nanometer accuracy and sub-millisecond time resolution. They are used to study a wide range of biological processes, from cellular motility to DNA replication and segregation. Key components include a trapping laser and a high numerical aperture (NA) microscope objective. Position detection methods, such as back focal plane (BFP) interferometry, are crucial for accurate measurements. Magnetic tweezers are simpler to implement and can exert forces up to 1 nN. They are particularly useful for studying nucleic acid enzymes, such as DNA topoisomerases, due to their ability to apply infinite bandwidth, passive force clamping over large displacements. Magnetic tweezers are selective and insensitive to sample preparation, making them suitable for complex, heterogeneous environments. AFM is another technique used in single-molecule force spectroscopy, offering high-resolution imaging and force measurements. It is particularly useful for studying the mechanical properties of biological macromolecules and the interactions between them. The choice of technique depends on the specific application, with each technique having its strengths and limitations. Optical tweezers are versatile but can be affected by sample heating and photodamage, while magnetic tweezers are selective and insensitive to sample preparation but have limited force and displacement ranges. AFM is highly resolving but more complex and expensive to set up. Overall, single-molecule force spectroscopy techniques have revolutionized the study of biological processes by providing precise and detailed measurements of forces and motions at the molecular level.