Gene expression regulation, in particular at the level of transcription, has been demonstrated to play a key role in the development of human diseases, including cancer, and in bacteria it is crucial for proliferation as well as for pathogenicity. Transcriptional regulation is based on complex networks of interactions, including those of the regulatory proteins with the operator DNAs, which are further modulated by ligands. Thus, understanding transcriptional regulation mechanisms requires a thorough analysis of the physical parameters underlying the interactions involved. Among the panoply of methods available, fluorescence spectroscopy–based approaches have been widely used for the assessment of the thermodynamics and structural dynamics of biomolecular interactions. Here we will discuss the application of three fluorescence spectroscopy methods—fluorescence anisotropy and fluorescence correlation and cross-correlation spectroscopy—for the investigation of protein–DNA, protein–protein, and protein–ligand interactions. The weaknesses and the strengths of each method will be highlighted on the basis of our experience in the analysis of the interactions of bacterial repressors implicated in transcriptional regulation in bacilli.