Fluorescence-based microscopy techniques are key tools for studying protein dynamics in vivo, enabling real-time tracking of molecular interactions with high specificity and subcellular resolution. Fluorescence lifetime imaging microscopy (FLIM) provides quantitative insights into the microenvironment of fluorophores by measuring their excited-state decay times, independent of intensity-based variations such as concentration and photobleaching. The phasor approach to FLIM simplifies lifetime analysis by mapping decay dynamics onto a two-dimensional plot, eliminating complex fitting procedures and allowing real-time visualization of heterogeneous fluorescence signals. This approach enhances the detection of subtle microenvironmental changes, facilitating the study of protein interactions. This chapter explores the application of FLIM in molecular interaction studies, with a focus on Förster resonance energy transfer (FRET). Combining FLIM with FRET (FRET-FLIM) enables precise quantification of energy transfer efficiency, overcoming the limitations of intensity-based FRET measurements. Additionally, integrating FLIM with stimulated emission depletion (FLIM-STED) super-resolution microscopy extends the spatial resolution beyond the diffraction limit, allowing for the nanoscale mapping of protein distributions and interactions. Together, these advanced techniques provide powerful tools for investigating dynamic cellular processes with high temporal and spatial resolution, offering new perspectives on protein function and biomolecular mechanisms in living systems.