The biological function of many macromolecules such as proteins and RNA are tightly coupled to their conformation and their conformational dynamic. For example, the correct folding of a protein into its native state is essential for its biological function. Misfolded or unfolded proteins are known to be either (partially) inactive or to even display toxic functionality as aggregation of misfolded proteins is associated with multiple neurodegenerative diseases. Studying how proteins fold correctly and undergo conformational changes to accomplish their biological function is therefore crucial to understand the underlying biological mechanism and how diseases arise. Single-molecule Force Spectroscopy (SMFS) represents an ideal tool to study these molecular phenomena because of their unique capability to isolate individual biomolecules and observe conformational transitions and unfolding processes as they happen in real-time.