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We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in-depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley Sons, Inc. Basic Protocol Ratiometric smFRET detection and analysis of IDPs Support Protocol 1 Fluorophore labeling of a protei