AbstractWe present polarization‐resolved fluorescence measurements of fluorescent molecular rotors 9‐(2‐carboxy‐2‐cyanovinyl)julolidine (CCVJ), 9‐(2,2‐dicyanovinyl)julolidine (DCVJ), and a meso‐substituted boron dipyrromethene (BODIPY‐C12). The photophysical properties of these molecules are highly dependent on the viscosity of the surrounding solvent. The relationship between their quantum yields and the viscosity of the surrounding medium is given by an equation first described and presented by Förster and Hoffmann and can be used to determine the microviscosity of the environment around a fluorophore. Herein we evaluate the applicability of molecular rotors as probes of apparent viscosity on a microscopic scale based on their viscosity dependent fluorescence depolarization. We develop a theoretical framework, combining the Förster–Hoffmann equation with the Perrin equation and compare the dynamic ranges and usable working regimes for these dyes in terms of utilising fluorescence anisotropy as a measure of viscosity. We present polarization‐resolved fluorescence spectra and steady‐state fluorescence anisotropy imaging data for measurements of intracellular viscosity. We find that the dynamic range for fluorescence anisotropy for CCVJ and DCVJ is significantly lower than that of BODIPY‐C12 in the viscosity range 0.6<η<600 cP. Moreover, using steady‐state anisotropy measurements to probe microviscosity in the low (<3 cP) viscosity regime, the molecular rotors can offer a better dynamic range in anisotropy compared with a rigid dye as a probe of microviscosity, and a higher total working dynamic range in terms of viscosity.