This review discusses the chemical and photophysical properties of commonly used fluorophores in chemical biology, highlighting their roles in biochemical and biological research. Small-molecule fluorescent probes are essential tools in chemical biology, with their utility based on a small set of modular "core" dyes. These dyes can be modified with various chemical groups to create a wide range of fluorescent probes. Understanding the properties of these dyes is crucial for selecting the appropriate fluorophore for a given application. The review covers the history of fluorescent dyes, starting with quinine, a natural product that was the first well-defined small-molecule fluorophore. It discusses the development of fluorometers and the use of dyes in biological experiments. The importance of fluorescence-based technologies in chemical biology is emphasized, and the review explores the properties of various fluorophores, including endogenous fluorophores, polycyclic aromatics, coumarins, quinolines, indoles, imidazoles, NBD, fluorescein, rhodamine, naphthoxanthene dyes, phenanthridines, BODIPY, cyanines, phthalocyanines, and oxazines. Each class of fluorophore is discussed in terms of their chemical and photophysical properties, their applications in biological research, and their utility in various experimental techniques. The review also highlights the importance of understanding the strengths and weaknesses of each dye class to effectively utilize them in chemical biology research. The review concludes by emphasizing the importance of continued development of new and improved fluorescent probes to advance the field of chemical biology.This review discusses the chemical and photophysical properties of commonly used fluorophores in chemical biology, highlighting their roles in biochemical and biological research. Small-molecule fluorescent probes are essential tools in chemical biology, with their utility based on a small set of modular "core" dyes. These dyes can be modified with various chemical groups to create a wide range of fluorescent probes. Understanding the properties of these dyes is crucial for selecting the appropriate fluorophore for a given application. The review covers the history of fluorescent dyes, starting with quinine, a natural product that was the first well-defined small-molecule fluorophore. It discusses the development of fluorometers and the use of dyes in biological experiments. The importance of fluorescence-based technologies in chemical biology is emphasized, and the review explores the properties of various fluorophores, including endogenous fluorophores, polycyclic aromatics, coumarins, quinolines, indoles, imidazoles, NBD, fluorescein, rhodamine, naphthoxanthene dyes, phenanthridines, BODIPY, cyanines, phthalocyanines, and oxazines. Each class of fluorophore is discussed in terms of their chemical and photophysical properties, their applications in biological research, and their utility in various experimental techniques. The review also highlights the importance of understanding the strengths and weaknesses of each dye class to effectively utilize them in chemical biology research. The review concludes by emphasizing the importance of continued development of new and improved fluorescent probes to advance the field of chemical biology.