The article provides an overview of small-molecule fluorescent probes, which are essential tools in chemical biology for visualizing biochemical and biological processes. It highlights the importance of understanding the major classes of small-molecule fluorophores and their properties. The authors discuss the history of fluorescence, from the discovery of quinine as the first well-defined small-molecule fluorophore to the development of modern fluorescent proteins like green fluorescent protein (GFP). They also cover the chemical and photophysical properties of various fluorophores, including polycyclic aromatics, coumarins, quinolines, indoles, imidazoles, NBD, fluorescein, rhodamines, naphthoxanthene dyes, phenanthridines, BODIPY, cyanines, phthalocyanines, and oxazines. Each class of fluorophores is described in terms of its spectral characteristics, chemical stability, and applications in labeling biomolecules, enzyme substrates, environmental indicators, and cellular stains. The article emphasizes the versatility and importance of these fluorophores in both basic and applied research, particularly in chemical biology.The article provides an overview of small-molecule fluorescent probes, which are essential tools in chemical biology for visualizing biochemical and biological processes. It highlights the importance of understanding the major classes of small-molecule fluorophores and their properties. The authors discuss the history of fluorescence, from the discovery of quinine as the first well-defined small-molecule fluorophore to the development of modern fluorescent proteins like green fluorescent protein (GFP). They also cover the chemical and photophysical properties of various fluorophores, including polycyclic aromatics, coumarins, quinolines, indoles, imidazoles, NBD, fluorescein, rhodamines, naphthoxanthene dyes, phenanthridines, BODIPY, cyanines, phthalocyanines, and oxazines. Each class of fluorophores is described in terms of its spectral characteristics, chemical stability, and applications in labeling biomolecules, enzyme substrates, environmental indicators, and cellular stains. The article emphasizes the versatility and importance of these fluorophores in both basic and applied research, particularly in chemical biology.