Fluorescence spectroscopy methods have been proved to be powerful tools for the quantitative investigation of homologous and heterologous interactions, rotational and translational diffusion, and structural dynamics of biological molecules in crowded media. In addition to their high sensitivity, these methods present the advantage that the selective fluorescent labeling of the biomolecules under study allows distinguishing them from the background species. Moreover, the recent development of biological applications of single molecule fluorescence micro-spectroscopy methods has opened the possibility of performing quantitative determinations inside cells. In the last decades, theoretical and experimental studies have demonstrated the possible influence of the high concentration of macromolecules within living systems, on the thermodynamics and kinetics of biological reactions. Therefore, there is a growing interest in quantitatively evaluating the interactions involving biomolecules in the natural environment in which they occur. Since this is not always feasible, experiments conducted in model crowded conditions, resembling physiological media, may contribute to reduce the gap between traditional in vitro and in vivo experiments. In this review we will discuss the application of some fluorescence spectroscopy approaches, for the identification and quantification of biological macromolecules and their functional interactions in model crowded conditions.