Fluorescence resonance energy transfer provides information about protein structure and dynamics. Single-molecule analysis can capture the information normally lost through ensemble averaging of heterogeneous and dynamic samples. Immobilization of single molecules, under conditions that retain their biological activity, allows for extended observation of the same molecule for tens of seconds. This can capture slow conformational transitions or protein binding and unbinding cycles. Using an open geometry for immobilization allows for direct observation of the response to changing solution conditions or adding ligands. Here we provide detailed methods for immobilization and observation of fluorescently labeled single proteins using total internal reflection microscopy that are widely applicable to the study of intrinsically disordered proteins.