RNA folding pathways can be complex and even include kinetic traps or misfolded intermediates that can be slow to resolve. Characterizing these pathways is critical to understanding how RNA molecules acquire their biological function. We have previously developed a novel approach to help characterize such misfolded intermediates. Laser-assisted single-molecule refolding (LASR) is a powerful technique that combines temperature-jump (T-jump) kinetics with single-molecule detection. In a typical LASR experiment, the temperature is rapidly increased and conformational dynamics are characterized, in real-time, at the single-molecule level using single-molecule fluorescence resonance energy transfer (smFRET). Here, we provide detailed protocols for performing LASR experiments including sample preparation, temperature calibration, and data analysis.