Quantitative fluorescence emission anisotropy microscopy reveals the organization of fluorescently labeled cellular components and allows their characterization in terms of changes in either rotational diffusion or homo-Förster's energy transfer characteristics in living cells. These properties provide insights into molecular organization, such as orientation, confinement, and oligomerization in situ. Here we elucidate how quantitative measurements of anisotropy using multiple microscope systems may be made by bringing out the main parameters that influence the quantification of fluorescence emission anisotropy. We focus on a variety of parameters that contribute to errors associated with the measurement of emission anisotropy in a microscope. These include the requirement for adequate photon counts for the necessary discrimination of anisotropy values, the influence of extinction ratios of the illumination source, the detector system, the role of numerical aperture, and excitation wavelength. All these parameters also affect the ability to capture the dynamic range of emission anisotropy necessary for quantifying its reduction due to homo-FRET and other processes. Finally, we provide easily implementable tests to assess whether homo-FRET is a cause for the observed emission depolarization.