Optical tweezers have proven a useful tool for exploring the structure and function of individual molecules, such as proteins, DNA, and RNA. The ability to unfold and refold biological molecules has provided novel insights that complement and go beyond traditional biochemical and structural approaches. With sophisticated optical tweezers instrumentation coming to the market, single-molecule stretching studies now have become feasible and available to a wide range of users. Therefore, a step-by-step protocol for stretching individual biomolecules utilizing a simple experimental geometry is timely and presented here. While we have taken the unfolding of an RNA structure held between two RNA/DNA hybrid handles as an example, the technical protocol should be readily applicable to other biomolecules and may serve as a starting point for more sophisticated experiments.