The study of protein-protein interactions is fundamental to understanding how actin-dependent processes are controlled through the regulation of actin-binding proteins by their interactors. FRET-FLIM (Förster resonance energy transfer-fluorescence lifetime imaging microscopy) is a sensitive bioimaging method to detect protein-protein interactions in living cells through measurement of FRET, facilitated by the interactions of fluorophore-tagged fusion protein. As a sensitive and noninvasive method for the spatiotemporal visualization of dynamic protein-protein interactions, FRET-FLIM holds several advantages over other methods of protein interaction assays. FRET-FLIM has been widely employed to characterize many plant protein interactions, including interactions between actin-regulatory proteins and their binding partners. As we increasingly understand the plant actin cytoskeleton to coordinate a diverse number of complex functions, the study of actin-regulatory proteins and their interactors becomes increasingly technically challenging. Sophisticated and sensitive in vivo methods such as FRET-FLIM are likely to be crucial to the study of protein-protein interactions as more complex and challenging hypotheses are addressed.