Structural analyses of large, complex noncoding RNAs continue to lag behind their rapid discovery and functional descriptions. Site-specifically incorporated, minimally invasive fluorescent probes such as 2-aminopurine (2AP) and pyrrolo-cytosine (PyC) have provided essential complementary information about local RNA structure, conformational dynamics, and interactions. Here I describe a protocol that benchmarks and correlates local RNA conformations with their respective fluorescence lifetimes, as a general technique that confers key advantages over fluorescence intensity-based methods. The observation that fluorescence lifetimes are more sensitive to local structures than sequence contexts suggests broad utility across diverse RNA and ribonucleoprotein systems.