Abstract <p>The fluorescence quenching or turn-off strategy is an emerging method for determination of fluorescent and non-fluorescent active principal ingredients (API). It is gaining a growing interest in advanced pharmaceutical applications due to its high sensitivity and selectivity. The present review represents a comprehensive and up-to-date overview of the quantitative determination of drugs by fluorescence quenching-based methods with a special focus on probes for the first time. The theoretical fundamentals of fluorescence quenching, including static and dynamic quenching as well as the inner filter effect are presented. Subsequently, the key analytical and methodological considerations of fluorescence quenching-based methods, including analytical parameters (probe, solvent, pH), instrumentation, analytical performance with emphasis on application of green and white analytical chemistry principles, are highlighted. Finally, the properties of fluorescent probes (organic molecules, inorganic elements, salts, complexes, enzyme-catalyzed products, nanomaterials, nanocomposites, and hybrid materials) and their analytical approaches for drug determination are systematically appraised and summarized with relevant analytical conditions and parameters of the methods. The review also highlighted the advances and challenges of fluorescence quenching-based analytical methods.</p> Graphical Abstract <p></p>