In this chapter, we present the basic physical principles of the fluorescence anisotropy imaging microscopy (FAIM) and its application to study FP-tagged protein dynamics and interaction in live cells. The Förster mechanism of electronic energy transfer can occur between like chromophores (homo-fluorescence resonance energy transfer, homo-FRET) inducing fluorescence depolarization and can be monitored by fluorescence anisotropy. The energy transfer rate is fast compared to the rotational time of proteins, and therefore its detection as a fast depolarization process in the fluorescence anisotropy can be easily discriminated from rotational motion. Quantitative analysis of fluorescence anisotropy decays provides information on structural parameters: distance between the two interacting chromophores and spatial orientation between the chromophores within dimeric proteins. Fluorescence anisotropy decay is not easy to measure in living cells under the microscope and the instrumentations are necessarily sophisticated. In contrast, any type of microscope can be used to measure the steady-state anisotropy. Interestingly, two-photon excitation steady-state FAIM is a powerful tool for qualitative analysis of macromolecule interactions in living cells and can be used easily for time-lapse homo-FRET.